Xiong Ding, Kai Nie, Lei Shi, Yong Zhang, Li Guan, Dan Zhang, Shunxiang Qi and Xuejun Ma J. Clin. Microbiol. 2014, 52(6):1862. DOI: 10.1128/JCM.03298-13. Published Ahead of Print 19 March 2014.
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Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription−Isothermal Multiple-Self-Matching-Initiated Amplification Assay
Xiong Ding,a,b Kai Nie,b Lei Shi,a Yong Zhang,b Li Guan,b Dan Zhang,b Shunxiang Qi,c Xuejun Mab School of Light Industry and Food Sciences, South China University of Technology, Guangzhou city, Guangdong, Chinaa; Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, Chinab; Institute for Viral Disease Control and Prevention, Hebei Center for Disease Control and Prevention, Shijiazhuang, Hebei, Chinac
Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-footand-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription–isothermal multiple-selfmatching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R2 values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R2 values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses.
H
and-foot-and-mouth disease (HFMD) is a childhood syndrome and is characterized by tiny blisters on the skin and oral mucosa together with fever and poor appetite. The two major causative agents of HFMD are human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). EV71-related HFMD commonly accounts for the fatal cases, due to severe complications, such as brainstem encephalitis and rapid fatal pulmonary edema, whereas CVA16-related HFMD usually presents with mild symptoms (1– 3). Since no vaccine or antiviral drugs are currently available to treat HFMD, the early and rapid detection of EV71 and CVA16 are critical for the prevention and control of HFMD infection. Presently, laboratory detection of EV71 and CVA16 includes traditional virus isolation, neutralization, and nucleic acid amplification techniques. It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4–6). Nucleic acid amplification techniques, such as reverse transcription-PCR (RT-PCR) (7–9) and reverse transcription-quantitative PCR (qRT-PCR) (10, 11) take less time (2 to 3 h) and are generally used for making a standard diagnosis due to a high detection limit. In China, a few commercial qRT-PCR-based diagnostic kits for EV71 and CVA16 are available and have been approved by the State Food and Drug Administration for HFMD pathogen surveillance. However, PCR-based RNA amplification techniques particularly depend on
sophisticated and expensive equipment, skilled technicians, or a tedious process, which limits their applications in the primary diagnostics setting and for field deployment. To achieve simple and rapid RNA detection, the isothermal nucleic acid amplification technique has advantages, such as loopmediated isothermal amplification (LAMP) by the use of reverse transcriptase together with DNA polymerase, namely, the RTLAMP assay (12, 13). The RT-LAMP assay enables the rapid detection of viral RNA (⬍1 h) in a cheap water bath at a constant temperature (60 to 65°C), with a 10-fold- or 100-fold-higher detection limit than that of conventional RT-PCR (14). Further-
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Received 24 November 2013 Returned for modification 24 December 2013 Accepted 12 March 2014 Published ahead of print 19 March 2014 Editor: Y.-W. Tang Address correspondence to Shunxiang Qi, [email protected], or Xuejun Ma, [email protected]. X.D., K.N., and L.S. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JCM.03298-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03298-13
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Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay
Improved Detection Limit in Rapid Detection by RT-IMSA
more, products can be detected with the RT-LAMP assay through various methods. Apart from traditional gel electrophoresis, the RT-LAMP products can be detected through spectrophotometric equipment to measure turbidity (15) or through direct a visual inspection of turbidity (16) and color changes (17–19). To date, several RT-LAMP-based detection methods for EV71 and CVA16 have been established in our laboratory and with other groups (18, 20–22). Although the RT-LAMP assay can achieve a high detection limit with the artificial plasmid templates, its detection limit is not adequate for assessing clinical specimens with a low viral load in the early phase of HFMD, leading to a certain false-negative diagnosis rate (23). In this study, we developed a novel reverse transcription–isothermal multiple-self-matching-initiated amplification (RTIMSA) assay to offer simple and rapid RNA detection with an improved detection limit. The RT-IMSA assay resembles the RTLAMP assay, achieving detection by use of a reverse transcriptase and a strand displacement DNA polymerase. As shown in Fig. 1A, a total of three pairs of primers are used in the RT-IMSA assay, including one pair of forward and reverse stem primers, SteF and SteR, and two pairs of hybrid nested primers (the outer forward and reverse hybrid-primers DsF and DsR, and the inner forward and reverse hybrid-primers FIT and RIT). Compared with that of
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the RT-LAMP assay (Fig. 1B) (12, 24, 25), the primer design for the RT-IMSA assay has three distinct features. First, the primers specifically recognize seven distinct regions of the target, whereas those of the RT-LAMP assay recognize eight. Second, the outer primers in the RT-IMSA assay are two hybrid primers united with the regions of the stem primers, whereas they are nonhybrid outer primers in the RT-LAMP assay. Third, in the RT-IMSA assay, the 5= ends of the forward and reverse inner hybrid primers are complementary, whereas they are noncomplementary in the RTLAMP assay. Due to these modifications, the RT-IMSA assay can typically generate multiple self-matching structures (SMSs), which are single-stranded DNA amplicons generated by the hybrid primers. The SMS possesses a self-matching function because its 3= end sequence is entirely complementary to the partial sequence of its downstream region, which is similar to the dumbbell structure in the RT-LAMP assay. By applying the RT-IMSA assay to detect EV71 and CVA16, we find their detection limits are distinctly improved, especially when testing clinical specimens. MATERIALS AND METHODS Viruses and clinical specimens. The EV71 subgenotype C4 isolate (strain FY17.08/AN/CHN/2008, GenBank accession no. EU703812) and CVA16 isolate (strain FY18/AN/CHN/2008, GenBank accession no. EU812514)
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FIG 1 Principle of the RT-IMSA assay versus principle of the RT-LAMP assay with regard to primer design and initial amplification step. (A) Primer design of RT-IMSA reaction. Six primers are used in the RT-IMSA assay, including two stem primers (SteF and SteR) and two pairs of nested hybrid-primers (two outer primers of DsF and DsR and two inner primers of FIT and RIT). The primers specifically recognize seven distinct regions of the target cDNA labeled F3, F2, F1, T, R1c, R2c, and R3c from the 5= end. The DsF and DsR primers consist of the F3 and R3 and F1c and R1c sequences, the FIT and RIT primers consist of F2 and R2 and Tc and T sequences, and the SteF and SteR primers are the R1c and F1c sequences, respectively. (B) Primer design of RT-LAMP reaction. If taking into account the loop primers, six primers are also required in the RT-LAMP assay, including the two outer primers F3 and B3, the two inner hybrid-primers FIP and BIP, and the two loop primers LoopF and LoopB. The primers recognize eight distinct regions labeled F3, F2, LF, F1, B1c, LBc, B2c, and B3c from the 5= end. (C) The initial step of RT-IMSA. For ease of explanation, DNA synthesis initiated from DsF, and FIT is set as the starting process (DNA synthesis proceeds with DsR and BIT in a similar manner). Horizontal straight lines with arrows represent the direction of primer elongation. Angled lines with arrows represent primers annealing to sites on the target. Arcs with arrows represent the self-matching function of two regions. In the step, four basic self-matching structures (SMS-1 to -4) with different lengths are generated. (D) The initial step of RT-LAMP assay. Only one basic self-matching structure, namely, the dumbbell structure, is generated. F, forward direction (green); R, reverse direction (red); c, complementary sequence (blue, forward complementary; yellow, reverse complementary).
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80°C for 2 min to terminate the reaction. The products were then electrophoresed in 3.0% agarose. Two visual RT-IMSA assays were established individually by adding (i) a 1.0-l hydroxynaphthol blue (HNB) dye (3.0 mmol/liter; Lemongreen, Shanghai, China) to the mixture before amplification, according to reported visual RT-LAMP assays using HNB dye (17–19), and (ii) a 1.0-l modified HNB (mHNB) dye that consisted of 1.5 mol HNB and 1,000⫻ GeneFinder (Zeesan Biotech, Xiamen, China) after amplification, termed visual RT-IMSA (HNB) and RT-IMSA (mHNB) assays, respectively. The GeneFinder is a nucleic acid dye that was used to establish visual detection in the RT-LAMP assay (27, 28). The amplifications were performed in a water bath under the conditions described above. The products were then electrophoresed in 3.0% agarose. Particularly, a 5.0-l product of visual CVA16 RT-IMSA (HNB) was digested by a restriction enzyme of HindIII (Invitrogen, USA) at 37°C overnight and then electrophoresed in 3.0% agarose. The site of HindIII was intentionally inserted into the inner region of the DsF (between F1c and F3) or FIT (between Tc and F2) primer, and this site was not found in the sequences of the template tested or in other primers. Evaluation of the RT-IMSA assays. RNAs extracted from the control and reference viruses were used as templates to test the specificities of the RT-IMSA assays, with 3 replicates at each template and a no-target control included. The linearity of quantification was evaluated using the 10-fold RNA copy panel (equal to 107 to 10 copies per reaction). The detection limit was determined by serial dilutions of lower numbers of copies (equal to 10,000, 5,000, 1,000, 500, 250, 100, 50, 25, 10, 5, and 1 copy per reaction) in 15 replicates, as described previously (29, 30). The optimized one-tube real-time RT-LAMP assays established in our laboratory (18, 23) were conducted as parallel tests, using the same amount and type of templates. Briefly, a 25-l RT-LAMP reaction mixture contained 12.5 l of 2⫻ isothermal reaction mixture, 1.0 l of Bst 2.0 DNA polymerase (8 U/l), 0.5 l of avian myeloblastosis virus reverse transcriptase (10 U/l), 1.0 l of each primer (F3 and B3, 5.0 pmol/l; FIP and BIP, 40.0 pmol/l; LoopF and LoopB, 20.0 pmol/l), 1.0 l of RNase-free water, and 4.0 l of the RNA template. The reaction mixture was incubated in the real-time turbidimeter at 63°C for 60 min. The linearity of quantification and probit analysis of the detection limit were all analyzed using the SPSS 19.0 (IBM, USA) software. Performance of RT-IMSA detection with clinical specimens. The clinical performances of the RT-IMSA assays for the detection of EV71 and CVA16 were evaluated with a total of 261 clinical specimens from patients suspected to have HFMD. The real-time RT-LAMP assays as described above were carried out simultaneously as parallel tests. Commercial qRT-PCR diagnostic kits for EV71, CVA16, and panenterovirus RNA (BioPerfectus Technologies, Jiangsu, China) approved by the State Food and Drug Administration of China were adopted as the standard tools for diagnosis in the IVDC of the Hebei CDC in China. The qRT-PCR was conducted in an ABI 7300 device (Applied Biosystems, USA), and the specimens with threshold cycle (CT) values not higher than 35 were defined as positive according to the manufacturer’s instructions. Discrepant detection results compared with those from the commercial diagnostic kits were further verified using nested RT-PCR assays, which were the recommended methods for HFMD pathogen surveillance used in the CDC of provincial and municipal regions in China, as described previously (31, 32). The products of nested RT-PCRs were then subjected to sequencing for confirmation with an ABI 3730 automated DNA sequencer (Applied Biosystems) (32).
RESULTS
Principle of RT-IMSA. The principle of RT-IMSA reaction can be divided into two major steps, the basic SMS-producing step and the cycling amplification step of SMSs. Figure 1C displays the basic SMS-producing step. Following is a simple description of the process. DNA synthesis starts from the DsF and FIT primers as the first step (DNA synthesis proceeding with DsR and RIT is in a
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were used as reference viruses. Field isolates of human enterovirus known to be genetically related to HFMD were used as control viruses to evaluate the specificities of the RT-IMSA assays. The control viruses included coxsackievirus group A serotypes (CVA1, -2, -4, -5, -6, -7, -9, -10, -11, -12, -13, -14, -17, -20, and -24), coxsackievirus group B serotypes (CVB1, -2, -3, -4, -5, and -6), and enteric cytopathic human orphan (ECHO) viruses (serotypes 3, 6, 11, and 19). The control and reference isolates were all obtained from the National Laboratory for Poliomyelitis (NLP), National Institute for Viral Disease Control and Prevention (IVDC), and the Chinese Center for Disease Control and Prevention (CDC). To test the clinical performances of the RT-IMSA assays, a total of 261 clinical specimens from patients (1 month to 11 years of age) suspected to be infected with HFMD in Hebei province in China were collected by staff members of the IVDC of the Hebei CDC in China from December 2012 to July 2013. This study was entirely approved by the local ethics committee. The parents or grandparents of each child in the study provided informed consent for sample collection. All the viruses and clinical specimens were stored at ⫺80°C until use. RNA extraction and the RNA-copy panels used. Total RNAs were extracted from 140 l of the control viruses, reference viruses, and clinical specimens using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The RNAs were eluted in a final volume of 50-l RNase-free water. The RNAs were stored at ⫺80°C until use. The RNA panel at the copy level was made by RNAs transcribed in vitro. Briefly, the cDNA of the VP1 gene of EV71 or CVA16 was first cloned into a pGEM-T vector (Promega, Madison, WI, USA). Next, the recombinant plasmids extracted were linearized by the SpeI digestion (TaKaRa, Dalian, China). The digestion products were both transcribed in vitro into RNAs and purified by a RioMAX large-scale RNA production system-T7 (Promega). Finally, the RNA panels with 10-fold concentrations ranging from 2.5 ⫻ 106 to 2.5 copies per l were prepared and stored at ⫺80°C until use. Primer design of RT-IMSA assay for the detection of EV71 and CVA16. The VP1 gene sequences at genome positions of nucleotides (nt) 2978 to 3248 on EV71 strain SZ/HK08-6 (GenBank accession no. GQ279370.1) and nt 2551 to 2894 on CVA16 strain SZ/HK08-7 (GenBank accession no. GQ279371.1) from the GenBank database (http: //www.ncbi.nlm.nih.gov/) were used to design the primers for the EV71 and CVA16 RT-IMSA assays, respectively. These sequence regions selected were highly conserved regions of the EV71 and CVA16 VP1 genes, which were used and evaluated previously in our laboratory for the primer designs for the RT-LAMP assay (4, 18, 23). The primer design for the RT-IMSA assay was mainly based on the rules for that of the RT-LAMP assay, with the aid of PrimerExplorer V4 (see http://primerexplorer.jp /elamp4.0.0/; Eiken, Kyoto, Japan). The primers were analyzed for specificity through a BLAST search of the GenBank nucleotide database (http: //www.ncbi.nlm.nih.gov/BLAST/). The primers were synthetized and high-pressure liquid chromatography (HPLC) purified by Sangon Biotech (Shanghai, China). The genomic positions and sequence information of the RT-IMSA and RT-LAMP assay primers used in the study are shown in the Table S1 in the supplemental material. Development of one-tube real-time and visual RT-IMSA assays. The optimized one-tube RT-IMSA reaction was performed in a 25-l mixture containing 12.5 l of 2⫻ isothermal reaction mixture (combination of reaction buffer and deoxynucleoside triphosphates [dNTPs]) purchased from Deaou Biotechnology (Guangzhou, China), 1.0 l of Bst 2.0 DNA polymerase (8 U/l; New England BioLabs, Ipswich, MA, USA), 0.5 l of avian myeloblastosis virus reverse transcriptase (10 U/l; Promega, Madison, WI, USA), 1.0 l of each primer (DsF and DsR, 5.0 pmol/l; FIT and RIT, 20.0 pmol/l; and SteF and SteR, 40.0 pmol/l), 1.0 l of RNase-free water, and 4.0 l of the RNA template. Like the real-time RT-LAMP assay using a turbidimeter (15, 26), the real-time RT-IMSA assay was also performed in an LA-320c Loopamp turbidimeter (Teramecs, Tokyo, Japan). The amplification was performed at 63°C for 60 min and then heated at
Improved Detection Limit in Rapid Detection by RT-IMSA
similar manner). When the cDNA of the RNA target is produced, the F2 region of the FIT primer anneals to the F2c site of the cDNA. If the F3 region of the DsF primer anneals to the F3c site, the DNA strand displacement occurs and causes the strand elongated from FIT replaced and released. Meanwhile, the DNA strand elongated from DsF is also produced, resulting in the generation of two distinct single strands. Subsequently, when the R2c and R3c sites on the two strands are annealed to by the R2 and R3 regions of the RIT and DsR primers, respectively, the strand displacement by DsR takes place. Next, four basic SMSs with different lengths (SMS-1 to SMS-4) are formed in this step. With the four SMSs as substrates, their cycling amplifications are initiated independently, the details of which can be seen in the Fig. S1 in the supplemental material. Visual RT-IMSA assays and the electrophoresis of amplified and digested products. For the visual RT-IMSA (HNB) assay, the tubes with positive reactions displayed a sky blue color, while the negative tubes displayed violet (Fig. 2A). For the visual RT-IMSA (mHNB) assay, the tubes with positive reactions displayed dark green, while the negative tubes displayed orange (Fig. 2B). The products of the positive reactions were all confirmed by electrophoresis. The product of the visual CVA16 RT-IMSA (HNB) assay
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was taken as an example. As shown in Fig. 3C, the positive RTIMSA reactions produced many bands with different sizes from bottom to top, and the number of these bands was distinctly more than that in the RT-LAMP assay when testing identical templates. The products of the visual CVA16 RT-IMSA (HNB) assay were then digested by a restriction enzyme of HindIII, the site of which was introduced in the DsF and FIT primers beforehand. Electrophoresis of the digested products indicated that four fragments were observed when the site was in the DsF primer, and three fragments were indicated when the site was in the FIT primer (Fig. 3D). For the RT-LAMP assay, however, only one fragment was observed with the same site inserted into the FIP primer (data not shown). The difference between the RT-IMSA and RT-LAMP assays in the electrophoresis of amplification and digestion products indirectly confirmed the generation of four basic SMSs in the RTIMSA assay. Specificities, linearity of quantification, and detection limits of the RT-IMSA assays. The real-time RT-IMSA assay for detecting EV71 and CVA16 possessed high specificity for the targets, as no cross-reactivity was observed with the RNA of any control virus. Similar results were observed for the visual RT-IMSA (HNB) and RT-IMSA (mHNB) assays, for which only the tubes
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FIG 2 Visual RT-IMSA assays and the electrophoresis of amplified and digested products. (A) Visual detection by RT-IMSA assay with the addition of HNB dye prior to amplification. Sky blue indicates positive reactions and violet indicates negative reactions. Tubes 1 to 4, EV71-positive, CVA16-positive, EV71-negative, and CVA16-negative, respectively. (B) Visual RT-IMSA by adding the modified HNB with a GeneFinder dye after amplification. The color of positive reaction was dark green, whereas the color of negative reactions was orange. Tubes 1 to 4, EV71-positive, CVA16-positive, EV71-negative, and CVA16-negative, respectively. (C) Electrophoresis of the products of visual RT-IMSA (HNB), including those of real-time RT-LAMP assay when testing identical templates (the VP1 RNA copies of CVA16). M, DL2000 marker; lanes 1 to 3, the products by RT-IMSA to amplify RNA copies of 103, 105, and 107, respectively; lanes 4 to 6, the products by RT-LAMP to amplify RNA copies of 103, 105, and 107, respectively. (D) Electrophoresis of digested products of visual CVA16 RT-IMSA (HNB) in which the site of HindIII restriction enzyme was introduced into the DsF and FIT primers. M, DL2000 marker; Lanes 1 to 4, the digested product of a positive reaction (the site in DsF), the digested product of positive (the site in FIT), the digested product of negative, and the amplified product of positive, respectively.
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with the target RNAs revealed sky blue and dark green colors, respectively. The linearity of quantification of the real-time RT-IMSA and RT-LAMP assays to test EV71 and CVA16 was established through a linear regression plot by plotting the time-to-positivity values against the values of log10 RNA copies tested per reaction. The EV71 RT-IMSA reached 103 copies/reaction with the linear correlations of an R2 value of 0.952, and the interassay coefficient of variation (CV) was from 1.493% to 6.045% (Fig. 3A); the EV71 RT-LAMP assay reached 104 with an R2 value of 0.803, and the CV was 0.773 to 3.833% (Fig. 3B). For the detection of CVA16, the RT-IMSA assay reached 102 with an R2 value of 0.967, and the CV was 1.962 to 5.296% (Fig. 3C); the RT-LAMP assay reached 103 with an R2 value of 0.904, and the CV was 1.962 to 6.132% (Fig. 3D). Thus, a better linearity of quantification was achieved in the real-time RT-IMSA assay. The detection limit was validated by testing multiple replicates of dilutions of the templates. Dilutions (equal to 10,000, 5,000, 1,000, 500, 250, 100, 50, 25, 10, 5, and 1 copies/reaction) were tested in batches of 3 replicates on 5 separate assay runs, giving a total of 15 replicates at each dilution (Table 1). Using probit analysis with SPSS 19.0, the number of positive results at each dilution
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TABLE 1 Assay data of established RT-IMSA assays and the real-time RT-LAMP for probit analysis No. of positive samples/no. of samples tested by indicated assays for detection of EV71 (CVA16)b Concn (copies/ reaction)a
Real-time RT-IMSA
Visual RT-IMSA (mHNB)
Visual RT-IMSA (HNB)
Real-time RT-LAMP
10,000 5,000 1,000 500 250 100 50 25 10 5 1
15 (15) 15 (15) 15 (15) 12 (15) 9 (15) 6 (15) 3 (12) 0 (9) 0 (3) 0 (3) 0 (0)
15 (15) 15 (15) 15 (15) 10 (15) 9 (15) 6 (15) 3 (9) 0 (6) 0 (3) 0 (0) 0 (0)
15 (15) 15 (15) 15 (15) 9 (15) 6 (15) 3 (15) 0 (9) 0 (3) 0 (0) 0 (0) 0 (0)
15 (15) 15 (15) 9 (15) 6 (15) 3 (12) 3 (6) 0 (3) 0 (0) 0 (0) 0 (0) 0 (0)
a
EV71 or CVA16 RNAs of VP1 gene transcribed in vitro were used as the templates. Each dilution was tested in batches of 3 replicates on 5 separate assay runs, giving a total of 15 replicates. b
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FIG 3 Linearity of quantification of the real-time RT-IMSA and RT-LAMP assays to test EV71 and CVA16 established through a linear regression plot by plotting the time-to-positivity values against the values of log10 RNA copies tested per reaction. Linearity of quantification of RT-IMSA for the detection of EV71 (A), linearity of quantification of RT-LAMP for the detection of EV71 (B), linearity of quantification of RT-IMSA for the detection of CVA16 (C), and linearity of quantification of RT-LAMP for the detection of CVA16. NTC, no-target control. OD, optical density.
Improved Detection Limit in Rapid Detection by RT-IMSA
DISCUSSION
Currently, due to HFMD remaining an important public health problem in China, a simple, specific, and sensitive diagnostic method or kit for the rapid detection of EV71 and CVA16 is in great demand, especially for the primary diagnostics setting in rural areas. In this regard, a novel one-tube RT-IMSA assay resembling the RT-LAMP assay was developed and evaluated for an improved detection limit in the detection of EV71 and CVA16. The novel RT-IMSA assay typically relies on cycling strand displacement DNA synthesis based on the four SMSs generated in
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the initial step, which is performed by a DNA polymerase with strong strand displacement activity and a set of four hybrid nested primers and two stem primers. Apart from the difference in primer designs, the RT-IMSA assay is also distinctly different from the RT-LAMP assay in other three aspects. First, in the initial step of the RT-IMSA assay, four pairs of basic SMSs with different lengths are generated, whereas in the RT-LAMP assay, only one pair of SMSs (namely, dumbbells) is generated during its initial step (Fig. 1D). Second, all six primers of the RT-IMSA assay execute their functions in both the initial and cycling steps, whereas in the RT-LAMP assay, the outer primers (F3 and B3) contribute to the initial step only (12, 24, 25). Third, the optimal concentration ratio of the three pairs of primers in the RT-IMSA assay is 1:4:8 (DsF/DsR to FIT/RIT to SteF/SteR), whereas in the RT-LAMP assay, the optimal primer ratio is 1:8:4 (F3/B3 to FIP/BIP to LoopF/LoopB). The RT-IMSA assay theoretically produces more amplicons than the RT-LAMP assay due to the introduction of four hybrid nested primers, which results in an increased detection limit. The assumption was confirmed practically through the detection of EV71 and CVA16 by the RT-IMSA assay. To make a fair comparison, the regions of the target sequences for primer design for the RT-LAMP assay were selected to design the primers of the RT-IMSA assay, and the optimal RT-IMSA assay was compared with the optimal RT-LAMP assay using the same amount and type of templates. Regarding the testing of the artificial templates of VP1 RNAs transcribed in vitro, the real-time RT-IMSA assay exhibits a better linearity of quantification and higher detection limit than the real-time RT-LAMP assay. Additionally, a higher detection limit was also achieved in the visual RT-IMSA assays we established. Although the visual RT-IMSA (HNB) assay has a lower detection limit than the visual RT-IMSA (mHNB) assay, the former, without opening tube caps, may reduce the risk of cross-contamination. Regarding the specificity of the test, the RT-IMSA assays have a high degree of specificity to the target RNA, which is related to the six primers that specifically recognize seven distinct regions on the target sequence. In order to test the clinical utility of the RT-IMSA assay, both the real-time and visual assays were further evaluated with a total of 261 specimens related to HFMD, and the qRT-PCR assay using commercial kits was conducted as a standard test. The results showed that EV71 and CVA16 were still the two major pathogens (71.3% [186/261]) causing the outbreak of HFMD in Hebei province from December 2012 to July 2013, but the spread of CVA16 (49.8% [130/261]) was larger than that of EV71 (21.5% [56/261]), showing a shift in the circulating trends of the two viruses, which was in accordance with the results of a new published report (33). In the evaluation, all the positive samples by our new approaches also tested positive by the corresponding qRT-PCRs. However, there were a few supposedly false-negative samples that were negative by the new approaches while positive by qRT-PCR. Regarding the detection of EV71, the numbers of false-negative samples were 2, 4, and 2 for the real-time RT-IMSA, visual RT-IMSA (HNB), and visual RT-IMSA (mHNB) assays, respectively; regarding CVA16 detection, the numbers were 7, 10, and 8, respectively (Table 2). Actually, these samples were found to be EV71 or CVA16 positive, with high CT values (⬎33) by qRT-PCR, which were further confirmed by nested RT-PCR and sequencing to be true positives. For the EV71- or CVA16-negative specimens that contained 70 other-enterovirus-positive samples, the diagnostic results from the new approaches and corresponding qRT-PCRs
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was used to calculate the EV71 or CVA16 RNA copies having a 95% probability of detection. For the detection of EV71 RNA copies, the 95% detection limit of the real-time RT-IMSA assay was approximately 937 copies/reaction, and the 95% confidence limit (CI) ranged from 621 to 1,555 copies/reaction; the detection limits of the visual RT-IMSA (HNB), visual RT-IMSA (mHNB), and real-time RT-LAMP assays were approximately 1,749 (CI, 1,140 to 2,967), 1,039 (CI, 690 to 1,722), and 3,266 (CI, 2,101 to 5,651) copies/reaction, respectively. Regarding the detection of CVA16 RNA copies, the detection limits of the real-time RTIMSA, visual RT-IMSA (HNB), visual RT-IMSA (mHNB), and real-time RT-LAMP assays were approximately 67 (CI, 48 to 102), 149 (CI, 105 to 228), 108 (CI, 77 to 164), and 430 (CI, 305 to 660) copies/reaction, respectively. The results indicated that both the real-time and visual RT-IMSA assays had higher detection limits than that of real-time RT-LAMP assay for detecting EV71 or CVA16 RNA copies. Evaluation assays with clinical specimens. As shown in Table 2, 21.5% (56/261), 49.8% (130/261), and 98.1% (256/261) of the specimens were found to be EV71-, CVA16-, and panenteroviruspositive, respectively, via the commercial qRT-PCR kits. Coinfection of EV71 and CVA16 was not found in the 261 specimens, and so, the number of both EV71- and CVA16-negative specimens was 75. Of the 75 specimens, 93.3% (70/75) were classified as other enteroviruses by the panenterovirus qRT-PCR kit. The remaining 5 samples were the negative samples, as no enterovirus was identified in these samples. In comparison with the qRT-PCR assay for detecting EV71, the sensitivities of the real-time RT-IMSA, visual RT-IMSA (HNB), visual RT-IMSA (mHNB), and real-time RT-LAMP assays were 96.4% (95% confidence limit [CI], 87.7 to 99.6%), 92.9% (95% CI, 82.7 to 98.0%), 96.4% (95% CI, 87.7 to 99.6%), and 91.1% (95% CI, 80.4 to 97.0%), respectively; for detecting CVA16, the sensitivities were 94.6% (95% CI, 89.2 to 97.8%), 92.3% (95% CI, 86.3 to 96.3%), 93.9% (95% CI, 88.2 to 97.3%), and 90.8% (95% CI, 84.4 to 95.1%), respectively (Table 2). The samples with discrepant detection results (qRT-PCR positive, with CT values of ⬎33 while testing negative with the RT-IMSA and RT-LAMP assays) were further confirmed by sequencing to be true positives. Therefore, our study demonstrated that the clinical performances of the real-time and visual RT-IMSA assays both displayed higher diagnostic sensitivities than that of real-time the RT-LAMP assay for detecting EV71 and CVA16. For the detection of 205 EV71negative specimens, which contained 70 other-enterovirus-positive samples, the results from new approaches and the qRT-PCR were in complete agreement, as were the results of the detection of 131 CVA16-negative specimens (Table 2). Consequently, 100% specificities were achieved by our new approaches, including when testing other-enteroviruses-positive specimens.
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54 0 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9%)
123 0 94.6 (89.2–97.8) 100.0 (97.2–100.0) 100.0 (97.1–100.0) 94.9 (89.8–97.9)
EV71 No. positive (56) No. negative (205)c Sensitivity Specificity Positive predictive value Negative predictive value
CVA16 No. positive (130) No. negative (131)d Sensitivity Specificity Positive predictive value Negative predictive value 7 131 94.6 (89.2–97.8) 100.0 (97.2–100.0) 100.0 (97.1–100.0) 94.9 (89.8–97.9)
2 205 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9%)
Negative
b
120 0 92.3 (86.3–96.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 92.9 (87.3–96.6)
52 0 92.9 (82.7–98.0) 100.0 (98.2–100.0) 100.0 (93.2–100.0) 98.1 (95.2–99.5)
Positive
b
10 131 92.3 (86.3–96.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 92.9 (87.3–96.6)
4 205 92.9 (82.7–98.0) 100.0 (98.2–100.0) 100.0 (93.2–100.0) 98.1 (95.2–99.5)
Negative
Visual RT-IMSA (HNB)
122 0 93.9 (88.2–97.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 94.2 (89.0–97.5)
54 0 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9)
Positive
b
8 131 93.9 (88.2–97.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 94.2 (89.0–97.5)
2 205 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9)
Negative
Visual RT-IMSA (mHNB)
118 0 90.8 (84.4–95.1) 100.0 (97.2–100.0) 100.0 (96.9–100.0) 91.6 (85.8–95.6)
51 0 91.1 (80.4–97.0) 100.0 (98.2–100.0) 100.0 (93.0–100.0) 97.6 (94.5–99.2)
Positive
Real-time RT-LAMP
12 131 90.8 (84.4–95.1) 100.0 (97.2–100.0) 100.0 (96.9–100.0) 94.2 (89.0–97.5)
5 205 91.1 (80.4–97.0) 100.0 (98.2–100.0) 100.0 (93.0–100.0) 97.6 (94.5–99.2)
Negativeb
a The commercial qRT-PCR diagnostic kits for EV71 and CVA16 were approved by the State Food and Drug Administration of China and adopted as a standard diagnosis in the IVDC of the Hebei CDC in China. The data are represented as the percentage (95 confidence interval [CI]) unless otherwise stated. A total of 261 clinical specimens from suspicious patients (age 1 month to 11 years old) with HFMD in Hebei province in China were collected from December 2012 to July 2013. One sample for both EV71- and CVA16-negative stool samples was used as a negative control. b The negative samples detected by RT-IMSA or RT-LAMP were found to be EV71 or CVA16 positive, with high CT values (⬎33) by qRT-PCR and were further confirmed by nested-RT-PCR and sequencing to be true positive. c A total of 205 EV71-negative specimens detected by qRT-PCR included 130 CVA16-positive, 70 panenterovirus-positive, and 5 negative samples. d A total of 131 CVA16-negative samples detected by qRT-PCR included 56 EV71-positive, 70 panenterovirus -positive, and 5 negative samples.
Positive
qRT-PCR results (n) by serotypea
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Real-time RT-IMSA
TABLE 2 Clinical performance of established RT-IMSA assays versus that of real-time RT-LAMP compared with a commercial qRT-PCR diagnostic kit to detect EV71 and CVA16 from clinical specimens
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Improved Detection Limit in Rapid Detection by RT-IMSA
ACKNOWLEDGMENTS This work was supported by the China Mega-Project for Infectious Disease (grants 2012ZX10004-215, 2013ZX10004-001, 2013ZX10004-202, and 2014ZX10004-003). We thank the Beijing IPE Biotechnology Co., Ltd., for the animation designs for the principle and software development for the primer design of the RT-IMSA. We declare no conflicts of interest.
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were in complete agreement, indicating 100% diagnostic specificities achieved by our new approaches even when testing otherenterovirus-positive specimens. Furthermore, in comparison to qRT-PCR, both the real-time and visual RT-IMSA assays had higher diagnostic sensitivities than that of the real-time RT-LAMP assay. The RT-IMSA reaction does not work with any one pair of primers unless at least two pairs of primers are required, but the reaction with three pairs of primers performs best for assay speed and detection limit (data not shown). Compared to the RT-LAMP assay, an improved detection limit was also obtained using the RT-IMSA technique to detect the infectious pathogen of influenza A (H7N9) virus, just as with the EV71 and CVA16 examples (our unpublished data). The software for the RT-IMSA primer design will soon be available to assist the scientific community in configuring the RT-IMSA assay. In conclusion, the novel RT-IMSA assay is superior to RT-LAMP assay in terms of the detection limit and has the potential to be widely used as a platform for the simple and rapid detection of EV71, CVA16, and other RNA viruses.
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Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription−Isothermal Multiple-Self-Matching-Initiated Amplification Assay
Xiong Ding,a,b Kai Nie,b Lei Shi,a Yong Zhang,b Li Guan,b Dan Zhang,b Shunxiang Qi,c Xuejun Mab School of Light Industry and Food Sciences, South China University of Technology, Guangzhou city, Guangdong, Chinaa; Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping district, Beijing, Chinab; Institute for Viral Disease Control and Prevention, Hebei Center for Disease Control and Prevention, Shijiazhuang, Hebei, Chinac
Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-footand-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription–isothermal multiple-selfmatching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R2 values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R2 values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses.
H
and-foot-and-mouth disease (HFMD) is a childhood syndrome and is characterized by tiny blisters on the skin and oral mucosa together with fever and poor appetite. The two major causative agents of HFMD are human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). EV71-related HFMD commonly accounts for the fatal cases, due to severe complications, such as brainstem encephalitis and rapid fatal pulmonary edema, whereas CVA16-related HFMD usually presents with mild symptoms (1– 3). Since no vaccine or antiviral drugs are currently available to treat HFMD, the early and rapid detection of EV71 and CVA16 are critical for the prevention and control of HFMD infection. Presently, laboratory detection of EV71 and CVA16 includes traditional virus isolation, neutralization, and nucleic acid amplification techniques. It is difficult to achieve rapid detection using traditional virus isolation and neutralization because of their low specificities and detection limits, as well as the long time that is spent obtaining results (4–6). Nucleic acid amplification techniques, such as reverse transcription-PCR (RT-PCR) (7–9) and reverse transcription-quantitative PCR (qRT-PCR) (10, 11) take less time (2 to 3 h) and are generally used for making a standard diagnosis due to a high detection limit. In China, a few commercial qRT-PCR-based diagnostic kits for EV71 and CVA16 are available and have been approved by the State Food and Drug Administration for HFMD pathogen surveillance. However, PCR-based RNA amplification techniques particularly depend on
sophisticated and expensive equipment, skilled technicians, or a tedious process, which limits their applications in the primary diagnostics setting and for field deployment. To achieve simple and rapid RNA detection, the isothermal nucleic acid amplification technique has advantages, such as loopmediated isothermal amplification (LAMP) by the use of reverse transcriptase together with DNA polymerase, namely, the RTLAMP assay (12, 13). The RT-LAMP assay enables the rapid detection of viral RNA (⬍1 h) in a cheap water bath at a constant temperature (60 to 65°C), with a 10-fold- or 100-fold-higher detection limit than that of conventional RT-PCR (14). Further-
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Journal of Clinical Microbiology
Received 24 November 2013 Returned for modification 24 December 2013 Accepted 12 March 2014 Published ahead of print 19 March 2014 Editor: Y.-W. Tang Address correspondence to Shunxiang Qi, [email protected], or Xuejun Ma, [email protected]. X.D., K.N., and L.S. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JCM.03298-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03298-13
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Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay
Improved Detection Limit in Rapid Detection by RT-IMSA
more, products can be detected with the RT-LAMP assay through various methods. Apart from traditional gel electrophoresis, the RT-LAMP products can be detected through spectrophotometric equipment to measure turbidity (15) or through direct a visual inspection of turbidity (16) and color changes (17–19). To date, several RT-LAMP-based detection methods for EV71 and CVA16 have been established in our laboratory and with other groups (18, 20–22). Although the RT-LAMP assay can achieve a high detection limit with the artificial plasmid templates, its detection limit is not adequate for assessing clinical specimens with a low viral load in the early phase of HFMD, leading to a certain false-negative diagnosis rate (23). In this study, we developed a novel reverse transcription–isothermal multiple-self-matching-initiated amplification (RTIMSA) assay to offer simple and rapid RNA detection with an improved detection limit. The RT-IMSA assay resembles the RTLAMP assay, achieving detection by use of a reverse transcriptase and a strand displacement DNA polymerase. As shown in Fig. 1A, a total of three pairs of primers are used in the RT-IMSA assay, including one pair of forward and reverse stem primers, SteF and SteR, and two pairs of hybrid nested primers (the outer forward and reverse hybrid-primers DsF and DsR, and the inner forward and reverse hybrid-primers FIT and RIT). Compared with that of
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the RT-LAMP assay (Fig. 1B) (12, 24, 25), the primer design for the RT-IMSA assay has three distinct features. First, the primers specifically recognize seven distinct regions of the target, whereas those of the RT-LAMP assay recognize eight. Second, the outer primers in the RT-IMSA assay are two hybrid primers united with the regions of the stem primers, whereas they are nonhybrid outer primers in the RT-LAMP assay. Third, in the RT-IMSA assay, the 5= ends of the forward and reverse inner hybrid primers are complementary, whereas they are noncomplementary in the RTLAMP assay. Due to these modifications, the RT-IMSA assay can typically generate multiple self-matching structures (SMSs), which are single-stranded DNA amplicons generated by the hybrid primers. The SMS possesses a self-matching function because its 3= end sequence is entirely complementary to the partial sequence of its downstream region, which is similar to the dumbbell structure in the RT-LAMP assay. By applying the RT-IMSA assay to detect EV71 and CVA16, we find their detection limits are distinctly improved, especially when testing clinical specimens. MATERIALS AND METHODS Viruses and clinical specimens. The EV71 subgenotype C4 isolate (strain FY17.08/AN/CHN/2008, GenBank accession no. EU703812) and CVA16 isolate (strain FY18/AN/CHN/2008, GenBank accession no. EU812514)
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FIG 1 Principle of the RT-IMSA assay versus principle of the RT-LAMP assay with regard to primer design and initial amplification step. (A) Primer design of RT-IMSA reaction. Six primers are used in the RT-IMSA assay, including two stem primers (SteF and SteR) and two pairs of nested hybrid-primers (two outer primers of DsF and DsR and two inner primers of FIT and RIT). The primers specifically recognize seven distinct regions of the target cDNA labeled F3, F2, F1, T, R1c, R2c, and R3c from the 5= end. The DsF and DsR primers consist of the F3 and R3 and F1c and R1c sequences, the FIT and RIT primers consist of F2 and R2 and Tc and T sequences, and the SteF and SteR primers are the R1c and F1c sequences, respectively. (B) Primer design of RT-LAMP reaction. If taking into account the loop primers, six primers are also required in the RT-LAMP assay, including the two outer primers F3 and B3, the two inner hybrid-primers FIP and BIP, and the two loop primers LoopF and LoopB. The primers recognize eight distinct regions labeled F3, F2, LF, F1, B1c, LBc, B2c, and B3c from the 5= end. (C) The initial step of RT-IMSA. For ease of explanation, DNA synthesis initiated from DsF, and FIT is set as the starting process (DNA synthesis proceeds with DsR and BIT in a similar manner). Horizontal straight lines with arrows represent the direction of primer elongation. Angled lines with arrows represent primers annealing to sites on the target. Arcs with arrows represent the self-matching function of two regions. In the step, four basic self-matching structures (SMS-1 to -4) with different lengths are generated. (D) The initial step of RT-LAMP assay. Only one basic self-matching structure, namely, the dumbbell structure, is generated. F, forward direction (green); R, reverse direction (red); c, complementary sequence (blue, forward complementary; yellow, reverse complementary).
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80°C for 2 min to terminate the reaction. The products were then electrophoresed in 3.0% agarose. Two visual RT-IMSA assays were established individually by adding (i) a 1.0-l hydroxynaphthol blue (HNB) dye (3.0 mmol/liter; Lemongreen, Shanghai, China) to the mixture before amplification, according to reported visual RT-LAMP assays using HNB dye (17–19), and (ii) a 1.0-l modified HNB (mHNB) dye that consisted of 1.5 mol HNB and 1,000⫻ GeneFinder (Zeesan Biotech, Xiamen, China) after amplification, termed visual RT-IMSA (HNB) and RT-IMSA (mHNB) assays, respectively. The GeneFinder is a nucleic acid dye that was used to establish visual detection in the RT-LAMP assay (27, 28). The amplifications were performed in a water bath under the conditions described above. The products were then electrophoresed in 3.0% agarose. Particularly, a 5.0-l product of visual CVA16 RT-IMSA (HNB) was digested by a restriction enzyme of HindIII (Invitrogen, USA) at 37°C overnight and then electrophoresed in 3.0% agarose. The site of HindIII was intentionally inserted into the inner region of the DsF (between F1c and F3) or FIT (between Tc and F2) primer, and this site was not found in the sequences of the template tested or in other primers. Evaluation of the RT-IMSA assays. RNAs extracted from the control and reference viruses were used as templates to test the specificities of the RT-IMSA assays, with 3 replicates at each template and a no-target control included. The linearity of quantification was evaluated using the 10-fold RNA copy panel (equal to 107 to 10 copies per reaction). The detection limit was determined by serial dilutions of lower numbers of copies (equal to 10,000, 5,000, 1,000, 500, 250, 100, 50, 25, 10, 5, and 1 copy per reaction) in 15 replicates, as described previously (29, 30). The optimized one-tube real-time RT-LAMP assays established in our laboratory (18, 23) were conducted as parallel tests, using the same amount and type of templates. Briefly, a 25-l RT-LAMP reaction mixture contained 12.5 l of 2⫻ isothermal reaction mixture, 1.0 l of Bst 2.0 DNA polymerase (8 U/l), 0.5 l of avian myeloblastosis virus reverse transcriptase (10 U/l), 1.0 l of each primer (F3 and B3, 5.0 pmol/l; FIP and BIP, 40.0 pmol/l; LoopF and LoopB, 20.0 pmol/l), 1.0 l of RNase-free water, and 4.0 l of the RNA template. The reaction mixture was incubated in the real-time turbidimeter at 63°C for 60 min. The linearity of quantification and probit analysis of the detection limit were all analyzed using the SPSS 19.0 (IBM, USA) software. Performance of RT-IMSA detection with clinical specimens. The clinical performances of the RT-IMSA assays for the detection of EV71 and CVA16 were evaluated with a total of 261 clinical specimens from patients suspected to have HFMD. The real-time RT-LAMP assays as described above were carried out simultaneously as parallel tests. Commercial qRT-PCR diagnostic kits for EV71, CVA16, and panenterovirus RNA (BioPerfectus Technologies, Jiangsu, China) approved by the State Food and Drug Administration of China were adopted as the standard tools for diagnosis in the IVDC of the Hebei CDC in China. The qRT-PCR was conducted in an ABI 7300 device (Applied Biosystems, USA), and the specimens with threshold cycle (CT) values not higher than 35 were defined as positive according to the manufacturer’s instructions. Discrepant detection results compared with those from the commercial diagnostic kits were further verified using nested RT-PCR assays, which were the recommended methods for HFMD pathogen surveillance used in the CDC of provincial and municipal regions in China, as described previously (31, 32). The products of nested RT-PCRs were then subjected to sequencing for confirmation with an ABI 3730 automated DNA sequencer (Applied Biosystems) (32).
RESULTS
Principle of RT-IMSA. The principle of RT-IMSA reaction can be divided into two major steps, the basic SMS-producing step and the cycling amplification step of SMSs. Figure 1C displays the basic SMS-producing step. Following is a simple description of the process. DNA synthesis starts from the DsF and FIT primers as the first step (DNA synthesis proceeding with DsR and RIT is in a
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were used as reference viruses. Field isolates of human enterovirus known to be genetically related to HFMD were used as control viruses to evaluate the specificities of the RT-IMSA assays. The control viruses included coxsackievirus group A serotypes (CVA1, -2, -4, -5, -6, -7, -9, -10, -11, -12, -13, -14, -17, -20, and -24), coxsackievirus group B serotypes (CVB1, -2, -3, -4, -5, and -6), and enteric cytopathic human orphan (ECHO) viruses (serotypes 3, 6, 11, and 19). The control and reference isolates were all obtained from the National Laboratory for Poliomyelitis (NLP), National Institute for Viral Disease Control and Prevention (IVDC), and the Chinese Center for Disease Control and Prevention (CDC). To test the clinical performances of the RT-IMSA assays, a total of 261 clinical specimens from patients (1 month to 11 years of age) suspected to be infected with HFMD in Hebei province in China were collected by staff members of the IVDC of the Hebei CDC in China from December 2012 to July 2013. This study was entirely approved by the local ethics committee. The parents or grandparents of each child in the study provided informed consent for sample collection. All the viruses and clinical specimens were stored at ⫺80°C until use. RNA extraction and the RNA-copy panels used. Total RNAs were extracted from 140 l of the control viruses, reference viruses, and clinical specimens using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The RNAs were eluted in a final volume of 50-l RNase-free water. The RNAs were stored at ⫺80°C until use. The RNA panel at the copy level was made by RNAs transcribed in vitro. Briefly, the cDNA of the VP1 gene of EV71 or CVA16 was first cloned into a pGEM-T vector (Promega, Madison, WI, USA). Next, the recombinant plasmids extracted were linearized by the SpeI digestion (TaKaRa, Dalian, China). The digestion products were both transcribed in vitro into RNAs and purified by a RioMAX large-scale RNA production system-T7 (Promega). Finally, the RNA panels with 10-fold concentrations ranging from 2.5 ⫻ 106 to 2.5 copies per l were prepared and stored at ⫺80°C until use. Primer design of RT-IMSA assay for the detection of EV71 and CVA16. The VP1 gene sequences at genome positions of nucleotides (nt) 2978 to 3248 on EV71 strain SZ/HK08-6 (GenBank accession no. GQ279370.1) and nt 2551 to 2894 on CVA16 strain SZ/HK08-7 (GenBank accession no. GQ279371.1) from the GenBank database (http: //www.ncbi.nlm.nih.gov/) were used to design the primers for the EV71 and CVA16 RT-IMSA assays, respectively. These sequence regions selected were highly conserved regions of the EV71 and CVA16 VP1 genes, which were used and evaluated previously in our laboratory for the primer designs for the RT-LAMP assay (4, 18, 23). The primer design for the RT-IMSA assay was mainly based on the rules for that of the RT-LAMP assay, with the aid of PrimerExplorer V4 (see http://primerexplorer.jp /elamp4.0.0/; Eiken, Kyoto, Japan). The primers were analyzed for specificity through a BLAST search of the GenBank nucleotide database (http: //www.ncbi.nlm.nih.gov/BLAST/). The primers were synthetized and high-pressure liquid chromatography (HPLC) purified by Sangon Biotech (Shanghai, China). The genomic positions and sequence information of the RT-IMSA and RT-LAMP assay primers used in the study are shown in the Table S1 in the supplemental material. Development of one-tube real-time and visual RT-IMSA assays. The optimized one-tube RT-IMSA reaction was performed in a 25-l mixture containing 12.5 l of 2⫻ isothermal reaction mixture (combination of reaction buffer and deoxynucleoside triphosphates [dNTPs]) purchased from Deaou Biotechnology (Guangzhou, China), 1.0 l of Bst 2.0 DNA polymerase (8 U/l; New England BioLabs, Ipswich, MA, USA), 0.5 l of avian myeloblastosis virus reverse transcriptase (10 U/l; Promega, Madison, WI, USA), 1.0 l of each primer (DsF and DsR, 5.0 pmol/l; FIT and RIT, 20.0 pmol/l; and SteF and SteR, 40.0 pmol/l), 1.0 l of RNase-free water, and 4.0 l of the RNA template. Like the real-time RT-LAMP assay using a turbidimeter (15, 26), the real-time RT-IMSA assay was also performed in an LA-320c Loopamp turbidimeter (Teramecs, Tokyo, Japan). The amplification was performed at 63°C for 60 min and then heated at
Improved Detection Limit in Rapid Detection by RT-IMSA
similar manner). When the cDNA of the RNA target is produced, the F2 region of the FIT primer anneals to the F2c site of the cDNA. If the F3 region of the DsF primer anneals to the F3c site, the DNA strand displacement occurs and causes the strand elongated from FIT replaced and released. Meanwhile, the DNA strand elongated from DsF is also produced, resulting in the generation of two distinct single strands. Subsequently, when the R2c and R3c sites on the two strands are annealed to by the R2 and R3 regions of the RIT and DsR primers, respectively, the strand displacement by DsR takes place. Next, four basic SMSs with different lengths (SMS-1 to SMS-4) are formed in this step. With the four SMSs as substrates, their cycling amplifications are initiated independently, the details of which can be seen in the Fig. S1 in the supplemental material. Visual RT-IMSA assays and the electrophoresis of amplified and digested products. For the visual RT-IMSA (HNB) assay, the tubes with positive reactions displayed a sky blue color, while the negative tubes displayed violet (Fig. 2A). For the visual RT-IMSA (mHNB) assay, the tubes with positive reactions displayed dark green, while the negative tubes displayed orange (Fig. 2B). The products of the positive reactions were all confirmed by electrophoresis. The product of the visual CVA16 RT-IMSA (HNB) assay
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was taken as an example. As shown in Fig. 3C, the positive RTIMSA reactions produced many bands with different sizes from bottom to top, and the number of these bands was distinctly more than that in the RT-LAMP assay when testing identical templates. The products of the visual CVA16 RT-IMSA (HNB) assay were then digested by a restriction enzyme of HindIII, the site of which was introduced in the DsF and FIT primers beforehand. Electrophoresis of the digested products indicated that four fragments were observed when the site was in the DsF primer, and three fragments were indicated when the site was in the FIT primer (Fig. 3D). For the RT-LAMP assay, however, only one fragment was observed with the same site inserted into the FIP primer (data not shown). The difference between the RT-IMSA and RT-LAMP assays in the electrophoresis of amplification and digestion products indirectly confirmed the generation of four basic SMSs in the RTIMSA assay. Specificities, linearity of quantification, and detection limits of the RT-IMSA assays. The real-time RT-IMSA assay for detecting EV71 and CVA16 possessed high specificity for the targets, as no cross-reactivity was observed with the RNA of any control virus. Similar results were observed for the visual RT-IMSA (HNB) and RT-IMSA (mHNB) assays, for which only the tubes
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FIG 2 Visual RT-IMSA assays and the electrophoresis of amplified and digested products. (A) Visual detection by RT-IMSA assay with the addition of HNB dye prior to amplification. Sky blue indicates positive reactions and violet indicates negative reactions. Tubes 1 to 4, EV71-positive, CVA16-positive, EV71-negative, and CVA16-negative, respectively. (B) Visual RT-IMSA by adding the modified HNB with a GeneFinder dye after amplification. The color of positive reaction was dark green, whereas the color of negative reactions was orange. Tubes 1 to 4, EV71-positive, CVA16-positive, EV71-negative, and CVA16-negative, respectively. (C) Electrophoresis of the products of visual RT-IMSA (HNB), including those of real-time RT-LAMP assay when testing identical templates (the VP1 RNA copies of CVA16). M, DL2000 marker; lanes 1 to 3, the products by RT-IMSA to amplify RNA copies of 103, 105, and 107, respectively; lanes 4 to 6, the products by RT-LAMP to amplify RNA copies of 103, 105, and 107, respectively. (D) Electrophoresis of digested products of visual CVA16 RT-IMSA (HNB) in which the site of HindIII restriction enzyme was introduced into the DsF and FIT primers. M, DL2000 marker; Lanes 1 to 4, the digested product of a positive reaction (the site in DsF), the digested product of positive (the site in FIT), the digested product of negative, and the amplified product of positive, respectively.
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with the target RNAs revealed sky blue and dark green colors, respectively. The linearity of quantification of the real-time RT-IMSA and RT-LAMP assays to test EV71 and CVA16 was established through a linear regression plot by plotting the time-to-positivity values against the values of log10 RNA copies tested per reaction. The EV71 RT-IMSA reached 103 copies/reaction with the linear correlations of an R2 value of 0.952, and the interassay coefficient of variation (CV) was from 1.493% to 6.045% (Fig. 3A); the EV71 RT-LAMP assay reached 104 with an R2 value of 0.803, and the CV was 0.773 to 3.833% (Fig. 3B). For the detection of CVA16, the RT-IMSA assay reached 102 with an R2 value of 0.967, and the CV was 1.962 to 5.296% (Fig. 3C); the RT-LAMP assay reached 103 with an R2 value of 0.904, and the CV was 1.962 to 6.132% (Fig. 3D). Thus, a better linearity of quantification was achieved in the real-time RT-IMSA assay. The detection limit was validated by testing multiple replicates of dilutions of the templates. Dilutions (equal to 10,000, 5,000, 1,000, 500, 250, 100, 50, 25, 10, 5, and 1 copies/reaction) were tested in batches of 3 replicates on 5 separate assay runs, giving a total of 15 replicates at each dilution (Table 1). Using probit analysis with SPSS 19.0, the number of positive results at each dilution
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TABLE 1 Assay data of established RT-IMSA assays and the real-time RT-LAMP for probit analysis No. of positive samples/no. of samples tested by indicated assays for detection of EV71 (CVA16)b Concn (copies/ reaction)a
Real-time RT-IMSA
Visual RT-IMSA (mHNB)
Visual RT-IMSA (HNB)
Real-time RT-LAMP
10,000 5,000 1,000 500 250 100 50 25 10 5 1
15 (15) 15 (15) 15 (15) 12 (15) 9 (15) 6 (15) 3 (12) 0 (9) 0 (3) 0 (3) 0 (0)
15 (15) 15 (15) 15 (15) 10 (15) 9 (15) 6 (15) 3 (9) 0 (6) 0 (3) 0 (0) 0 (0)
15 (15) 15 (15) 15 (15) 9 (15) 6 (15) 3 (15) 0 (9) 0 (3) 0 (0) 0 (0) 0 (0)
15 (15) 15 (15) 9 (15) 6 (15) 3 (12) 3 (6) 0 (3) 0 (0) 0 (0) 0 (0) 0 (0)
a
EV71 or CVA16 RNAs of VP1 gene transcribed in vitro were used as the templates. Each dilution was tested in batches of 3 replicates on 5 separate assay runs, giving a total of 15 replicates. b
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FIG 3 Linearity of quantification of the real-time RT-IMSA and RT-LAMP assays to test EV71 and CVA16 established through a linear regression plot by plotting the time-to-positivity values against the values of log10 RNA copies tested per reaction. Linearity of quantification of RT-IMSA for the detection of EV71 (A), linearity of quantification of RT-LAMP for the detection of EV71 (B), linearity of quantification of RT-IMSA for the detection of CVA16 (C), and linearity of quantification of RT-LAMP for the detection of CVA16. NTC, no-target control. OD, optical density.
Improved Detection Limit in Rapid Detection by RT-IMSA
DISCUSSION
Currently, due to HFMD remaining an important public health problem in China, a simple, specific, and sensitive diagnostic method or kit for the rapid detection of EV71 and CVA16 is in great demand, especially for the primary diagnostics setting in rural areas. In this regard, a novel one-tube RT-IMSA assay resembling the RT-LAMP assay was developed and evaluated for an improved detection limit in the detection of EV71 and CVA16. The novel RT-IMSA assay typically relies on cycling strand displacement DNA synthesis based on the four SMSs generated in
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the initial step, which is performed by a DNA polymerase with strong strand displacement activity and a set of four hybrid nested primers and two stem primers. Apart from the difference in primer designs, the RT-IMSA assay is also distinctly different from the RT-LAMP assay in other three aspects. First, in the initial step of the RT-IMSA assay, four pairs of basic SMSs with different lengths are generated, whereas in the RT-LAMP assay, only one pair of SMSs (namely, dumbbells) is generated during its initial step (Fig. 1D). Second, all six primers of the RT-IMSA assay execute their functions in both the initial and cycling steps, whereas in the RT-LAMP assay, the outer primers (F3 and B3) contribute to the initial step only (12, 24, 25). Third, the optimal concentration ratio of the three pairs of primers in the RT-IMSA assay is 1:4:8 (DsF/DsR to FIT/RIT to SteF/SteR), whereas in the RT-LAMP assay, the optimal primer ratio is 1:8:4 (F3/B3 to FIP/BIP to LoopF/LoopB). The RT-IMSA assay theoretically produces more amplicons than the RT-LAMP assay due to the introduction of four hybrid nested primers, which results in an increased detection limit. The assumption was confirmed practically through the detection of EV71 and CVA16 by the RT-IMSA assay. To make a fair comparison, the regions of the target sequences for primer design for the RT-LAMP assay were selected to design the primers of the RT-IMSA assay, and the optimal RT-IMSA assay was compared with the optimal RT-LAMP assay using the same amount and type of templates. Regarding the testing of the artificial templates of VP1 RNAs transcribed in vitro, the real-time RT-IMSA assay exhibits a better linearity of quantification and higher detection limit than the real-time RT-LAMP assay. Additionally, a higher detection limit was also achieved in the visual RT-IMSA assays we established. Although the visual RT-IMSA (HNB) assay has a lower detection limit than the visual RT-IMSA (mHNB) assay, the former, without opening tube caps, may reduce the risk of cross-contamination. Regarding the specificity of the test, the RT-IMSA assays have a high degree of specificity to the target RNA, which is related to the six primers that specifically recognize seven distinct regions on the target sequence. In order to test the clinical utility of the RT-IMSA assay, both the real-time and visual assays were further evaluated with a total of 261 specimens related to HFMD, and the qRT-PCR assay using commercial kits was conducted as a standard test. The results showed that EV71 and CVA16 were still the two major pathogens (71.3% [186/261]) causing the outbreak of HFMD in Hebei province from December 2012 to July 2013, but the spread of CVA16 (49.8% [130/261]) was larger than that of EV71 (21.5% [56/261]), showing a shift in the circulating trends of the two viruses, which was in accordance with the results of a new published report (33). In the evaluation, all the positive samples by our new approaches also tested positive by the corresponding qRT-PCRs. However, there were a few supposedly false-negative samples that were negative by the new approaches while positive by qRT-PCR. Regarding the detection of EV71, the numbers of false-negative samples were 2, 4, and 2 for the real-time RT-IMSA, visual RT-IMSA (HNB), and visual RT-IMSA (mHNB) assays, respectively; regarding CVA16 detection, the numbers were 7, 10, and 8, respectively (Table 2). Actually, these samples were found to be EV71 or CVA16 positive, with high CT values (⬎33) by qRT-PCR, which were further confirmed by nested RT-PCR and sequencing to be true positives. For the EV71- or CVA16-negative specimens that contained 70 other-enterovirus-positive samples, the diagnostic results from the new approaches and corresponding qRT-PCRs
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was used to calculate the EV71 or CVA16 RNA copies having a 95% probability of detection. For the detection of EV71 RNA copies, the 95% detection limit of the real-time RT-IMSA assay was approximately 937 copies/reaction, and the 95% confidence limit (CI) ranged from 621 to 1,555 copies/reaction; the detection limits of the visual RT-IMSA (HNB), visual RT-IMSA (mHNB), and real-time RT-LAMP assays were approximately 1,749 (CI, 1,140 to 2,967), 1,039 (CI, 690 to 1,722), and 3,266 (CI, 2,101 to 5,651) copies/reaction, respectively. Regarding the detection of CVA16 RNA copies, the detection limits of the real-time RTIMSA, visual RT-IMSA (HNB), visual RT-IMSA (mHNB), and real-time RT-LAMP assays were approximately 67 (CI, 48 to 102), 149 (CI, 105 to 228), 108 (CI, 77 to 164), and 430 (CI, 305 to 660) copies/reaction, respectively. The results indicated that both the real-time and visual RT-IMSA assays had higher detection limits than that of real-time RT-LAMP assay for detecting EV71 or CVA16 RNA copies. Evaluation assays with clinical specimens. As shown in Table 2, 21.5% (56/261), 49.8% (130/261), and 98.1% (256/261) of the specimens were found to be EV71-, CVA16-, and panenteroviruspositive, respectively, via the commercial qRT-PCR kits. Coinfection of EV71 and CVA16 was not found in the 261 specimens, and so, the number of both EV71- and CVA16-negative specimens was 75. Of the 75 specimens, 93.3% (70/75) were classified as other enteroviruses by the panenterovirus qRT-PCR kit. The remaining 5 samples were the negative samples, as no enterovirus was identified in these samples. In comparison with the qRT-PCR assay for detecting EV71, the sensitivities of the real-time RT-IMSA, visual RT-IMSA (HNB), visual RT-IMSA (mHNB), and real-time RT-LAMP assays were 96.4% (95% confidence limit [CI], 87.7 to 99.6%), 92.9% (95% CI, 82.7 to 98.0%), 96.4% (95% CI, 87.7 to 99.6%), and 91.1% (95% CI, 80.4 to 97.0%), respectively; for detecting CVA16, the sensitivities were 94.6% (95% CI, 89.2 to 97.8%), 92.3% (95% CI, 86.3 to 96.3%), 93.9% (95% CI, 88.2 to 97.3%), and 90.8% (95% CI, 84.4 to 95.1%), respectively (Table 2). The samples with discrepant detection results (qRT-PCR positive, with CT values of ⬎33 while testing negative with the RT-IMSA and RT-LAMP assays) were further confirmed by sequencing to be true positives. Therefore, our study demonstrated that the clinical performances of the real-time and visual RT-IMSA assays both displayed higher diagnostic sensitivities than that of real-time the RT-LAMP assay for detecting EV71 and CVA16. For the detection of 205 EV71negative specimens, which contained 70 other-enterovirus-positive samples, the results from new approaches and the qRT-PCR were in complete agreement, as were the results of the detection of 131 CVA16-negative specimens (Table 2). Consequently, 100% specificities were achieved by our new approaches, including when testing other-enteroviruses-positive specimens.
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54 0 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9%)
123 0 94.6 (89.2–97.8) 100.0 (97.2–100.0) 100.0 (97.1–100.0) 94.9 (89.8–97.9)
EV71 No. positive (56) No. negative (205)c Sensitivity Specificity Positive predictive value Negative predictive value
CVA16 No. positive (130) No. negative (131)d Sensitivity Specificity Positive predictive value Negative predictive value 7 131 94.6 (89.2–97.8) 100.0 (97.2–100.0) 100.0 (97.1–100.0) 94.9 (89.8–97.9)
2 205 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9%)
Negative
b
120 0 92.3 (86.3–96.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 92.9 (87.3–96.6)
52 0 92.9 (82.7–98.0) 100.0 (98.2–100.0) 100.0 (93.2–100.0) 98.1 (95.2–99.5)
Positive
b
10 131 92.3 (86.3–96.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 92.9 (87.3–96.6)
4 205 92.9 (82.7–98.0) 100.0 (98.2–100.0) 100.0 (93.2–100.0) 98.1 (95.2–99.5)
Negative
Visual RT-IMSA (HNB)
122 0 93.9 (88.2–97.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 94.2 (89.0–97.5)
54 0 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9)
Positive
b
8 131 93.9 (88.2–97.3) 100.0 (97.2–100.0) 100.0 (97.0–100.0) 94.2 (89.0–97.5)
2 205 96.4 (87.7–99.6) 100.0 (98.2–100.0) 100.0 (93.4–100.0) 99.0 (96.6–99.9)
Negative
Visual RT-IMSA (mHNB)
118 0 90.8 (84.4–95.1) 100.0 (97.2–100.0) 100.0 (96.9–100.0) 91.6 (85.8–95.6)
51 0 91.1 (80.4–97.0) 100.0 (98.2–100.0) 100.0 (93.0–100.0) 97.6 (94.5–99.2)
Positive
Real-time RT-LAMP
12 131 90.8 (84.4–95.1) 100.0 (97.2–100.0) 100.0 (96.9–100.0) 94.2 (89.0–97.5)
5 205 91.1 (80.4–97.0) 100.0 (98.2–100.0) 100.0 (93.0–100.0) 97.6 (94.5–99.2)
Negativeb
a The commercial qRT-PCR diagnostic kits for EV71 and CVA16 were approved by the State Food and Drug Administration of China and adopted as a standard diagnosis in the IVDC of the Hebei CDC in China. The data are represented as the percentage (95 confidence interval [CI]) unless otherwise stated. A total of 261 clinical specimens from suspicious patients (age 1 month to 11 years old) with HFMD in Hebei province in China were collected from December 2012 to July 2013. One sample for both EV71- and CVA16-negative stool samples was used as a negative control. b The negative samples detected by RT-IMSA or RT-LAMP were found to be EV71 or CVA16 positive, with high CT values (⬎33) by qRT-PCR and were further confirmed by nested-RT-PCR and sequencing to be true positive. c A total of 205 EV71-negative specimens detected by qRT-PCR included 130 CVA16-positive, 70 panenterovirus-positive, and 5 negative samples. d A total of 131 CVA16-negative samples detected by qRT-PCR included 56 EV71-positive, 70 panenterovirus -positive, and 5 negative samples.
Positive
qRT-PCR results (n) by serotypea
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TABLE 2 Clinical performance of established RT-IMSA assays versus that of real-time RT-LAMP compared with a commercial qRT-PCR diagnostic kit to detect EV71 and CVA16 from clinical specimens
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Improved Detection Limit in Rapid Detection by RT-IMSA
ACKNOWLEDGMENTS This work was supported by the China Mega-Project for Infectious Disease (grants 2012ZX10004-215, 2013ZX10004-001, 2013ZX10004-202, and 2014ZX10004-003). We thank the Beijing IPE Biotechnology Co., Ltd., for the animation designs for the principle and software development for the primer design of the RT-IMSA. We declare no conflicts of interest.
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were in complete agreement, indicating 100% diagnostic specificities achieved by our new approaches even when testing otherenterovirus-positive specimens. Furthermore, in comparison to qRT-PCR, both the real-time and visual RT-IMSA assays had higher diagnostic sensitivities than that of the real-time RT-LAMP assay. The RT-IMSA reaction does not work with any one pair of primers unless at least two pairs of primers are required, but the reaction with three pairs of primers performs best for assay speed and detection limit (data not shown). Compared to the RT-LAMP assay, an improved detection limit was also obtained using the RT-IMSA technique to detect the infectious pathogen of influenza A (H7N9) virus, just as with the EV71 and CVA16 examples (our unpublished data). The software for the RT-IMSA primer design will soon be available to assist the scientific community in configuring the RT-IMSA assay. In conclusion, the novel RT-IMSA assay is superior to RT-LAMP assay in terms of the detection limit and has the potential to be widely used as a platform for the simple and rapid detection of EV71, CVA16, and other RNA viruses.
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