Arch Virol DOI 10.1007/s00705-014-2217-x
BRIEF REPORT
Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses D. Cecilia • M. Kakade • K. Alagarasu J. Patil • A. Salunke • D. Parashar • P. S. Shah
•
Received: 23 May 2014 / Accepted: 26 August 2014 Ó Springer-Verlag Wien 2014
Abstract Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaqueforming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate. Keywords Dengue chikungunya multiplex real time RT-PCR assay diagnosis Dengue virus (DENV; family Flaviviridae, genus Flavivirus) and chikungunya virus (CHIKV; family Togaviridae, genus Alphavirus) are transmitted by Aedes aegypti and Aedes albopictus mosquitoes and share the same geographic niche [1]. In the initial phase of infection, it is difficult to differentiate the two infections clinically. Whilst dengue can progress to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), CHIKV causes debilitating arthralgia, which may persist for months. Cocirculation and co-infections of CHIKV and DENV in India, Sri Lanka, Gabon, Cameroon, Madagascar,
D. Cecilia (&) M. Kakade K. Alagarasu J. Patil A. Salunke D. Parashar P. S. Shah Dengue/Chikungunya group, National Institute of Virology, Pune 411001, Maharashtra, India e-mail: [email protected]
Malaysia, Indonesia, Singapore, and Thailand have been recorded [2–5]. Differential diagnosis in these circumstances would help greatly in disease management. IgM against these viruses persists for months, and leftover IgM from a previous infection may lead to the wrong conclusion; therefore, serological methods have limitations [1]. A duplex conventional RT-PCR assay for distinguishing DENV and CHIKV [6] and a multiplex real-time RT-PCR have been reported earlier [7]. We had previously reported a two-step real-time RT-PCR for detection and quantification of DENV in human samples [8]. In the present study, we modified the assay to a one-step multiplex realtime RT-PCR assay for simultaneous detection of DENV and CHIKV. The National Institute of Virology (NIV) is a WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research and Rapid Diagnosis of Viral Diseases and routinely receives samples for diagnosis. Clinical samples that were left over after outbreak investigations were used in the study. This study was approved by the Human Ethics Committee of NIV. Waiver of the informed consent was granted by the committee on the basis of ‘‘Use of leftover specimens after clinical investigation’’ under the Indian Council of Medical Research Guidelines 2006. Prototype viruses were obtained from the virus repository of NIV. Stocks of DENV-1 (16007-genotype II, Thailand), DENV-2 (803347- genotype V, India), DENV-3 (633798 genotype I -USA), and DENV-4 (642069-genotype I, Thailand) were made in C6/36 (Aedes albopictus) cells, and CHIKV (062737- east/central/ south African (ECSA) genotype, India) in VERO (African green monkey kidney) cells. PS (porcine kidney stable) cells were used for virus titration by plaque-forming-unit (PFU) assay. PS and VERO cells were maintained in Dulbecco’s modified
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D. Cecilia et al. Table 1 List of primers and probes Primer/probe
Sequence
Genome position (accession number)
DENV-1,2,3 forward primer
GAR AGA CCA GAG ATC CTG CTG TCT
DENV1- 10647-10670 (KJ438296.1) DENV2- 10635-10658 (JX073928.1) DENV3- 10619-10642 (KJ160505.1)
DENV-1,2,3 reverse primer
ACC ATT CCA TTT TCT GGC GTT
DENV1- 10714-10694 (AY277666.2) DENV2- 10702-10682 (JX073928.1) DENV3- 10686-10666 (KF824903.1)
DENV-1,2,3 probe
AGC ATC ATT CCA GGC AC
DENV1- 10675-10691 (AY277666.2) DENV2- 10654-10670 (KJ189311.1) DENV3- 10647-10663 (KF824902.1)
DENV-4 forward primer
AAG CCA GGA GGA AGC TGT ACT CCT
10452-10475 (JQ513345.1)
DENV-4 reverse primer DENV-4 probe
CAA TCC ATC TTG CGG CGC TCT CTG TCT CTG CAA CAT CAA TCC AGG CA
10603-10583 (JQ513344.1) 10538-10563 (KF955510.1)
CHIKV forward primer
CGA AAA RGA RCC GGA GRA A
8401-8419 (JN558836.1)
CHIKV reverse primer
GAT AGT ACC CRG GKC TCA TGA CGT T
8465-8441 (JN558835.1)
CHIKV probe
CCC TRC GCA TGC TTG A
8421-8436 (JN558836.1)
Beta-actin forward primer
GGC ACC CAG CAC AAT GAA G
1044-1062 (NM_001101.3)
Beta-actin reverse primer
GCC GAT CCA CAC GGA GTA CT
1110-1091 (NM_001101.3)
Beta-actin probe
TCA AGA TCA TTG CTC CTC CTG AGA GCG C
1064-1086 (NM_001101.3)
Eagle medium (GIBCO BRL), and C6/36 in L15 (HIMEDIA). Medium was supplemented with 5 % foetal bovine serum and antibiotics. PS and VERO cultures were maintained at 37 °C and C6/36 cells at 28 °C with 5 % CO2. The viral RNA from clinical samples or cell culture fluids was extracted using a viral RNA purification kit (QIAmp Viral RNA Mini Kit, QIAGEN). Conventional RT-PCR for detection of CHIKV and serotyping of DENV were carried out as described earlier [9, 10]. The one-step multiplex real-time RT-PCR assay for DENV/CHIKV (DENV/CHIKV assay) was developed using primers and probes for DENV-1/2/3, DENV-4, CHIKV and beta-actin, which have been reported earlier [8, 11–13]. The primers and probes were targeted to regions specific to the different viruses but conserved among the genotypes. The sequences of primers and probes, along with GenBank accession numbers of the most similar sequence determined by BLAST analysis, are given in Table 1. The assay used TaqMan chemistry with minor groove binder (MGB) probes. The TaqMan probes were labeled with FAM (6carboxyfluorescein) for DENV-1/2/3, Cy5 (cyanine 5) for DENV-4, VIC (4,7,20 -trichloro-70 -phenyl-6-carboxyfluorescein) for CHIKV, and NED for beta-actin at the 50 end and BHQ-1/2 (Black Hole Quencher), a non-fluorescent quencher at the 30 end. The internal control reaction for beta-actin was carried out in a separate tube. All primers and probes were diluted to 10 pmoles/ll. The reaction mix included 0.875 ll of nuclease-free water, 5 ll
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of RNA, 12.5 ll of 2x RT-PCR buffer, 1 ll of 25X RTPCR enzyme (AgPath-ID one-step RT-PCR Kit; Applied Biosystems), 0.5 ll of three forward primers, 0.75 ll of three reverse primers and 0.625 ll of three probes specific for DENV-1/2/3, DENV-4 and CHIKV. The reaction was carried out using an ABI 7500 Real Time PCR system. The standard cycling conditions were used, which included an initial reverse transcription at 45 °C for 10 min, inactivation of reverse transcriptase at 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and annealing at 60 °C for 1 min. The detector was set to acquire emissions through FAM, Cy5 and VIC channels for each well. Threshold values were set above the background signal for each probe. On the basis of the nonspecific signal observed in non-template controls, a cutoff of cycle threshold (Ct) of 32 was decided. Samples that gave a Ct value of [32 were considered negative. For the preparation of viral RNA standards, RNA was extracted from virus stocks of DENV-3, DENV-4 and CHIKV, and the target region was amplified by RT-PCR and cloned into the TEasy cloning vector (Promega). The presence and orientation of the insert were confirmed by sequencing using the M13 primer. The plasmid was linearized by digestion with ApaI, and the target sequence was amplified using an in vitro RNA transcription kit (Roche Diagnostics). The in vitro-transcribed RNA was treated with DNase to digest the plasmid, and RNA was purified using a QIAamp Viral RNA Purification Kit (QIAGEN). The concentration of RNA was determined by spectrophotometry. The copy number of the RNA was calculated
Assay for detection of dengue and chikungunya viruses
based on the concentration of RNA and the molecular weight of each amplified target. The amplicons for DENV1, 2 and 3 were of similar length, and therefore the standard RNA of DENV-3 was used to calculate the viral load for all three viruses. Each of the primer/probe sets was tested in a monoplex real-time RT-PCR assay with RNA extracted from the prototype virus stocks containing 104 to 105 PFU. The Ct values ranged from 10 to 14. The primers and probes were then tested in the DENV/CHIKV assay using normal human serum spiked with 103 to 105 PFU of DENV-1, 2, 3, 4 or CHIKV. A positive signal was detected for FAM, representing DENV-1 (Ct = 18), DENV-2 (Ct = 14.2), DENV-3 (Ct = 18.2), for Cy5, representing DENV-4 (Ct = 21), and VIC, representing CHIKV (Ct = 12.6). The Ct values for NED, representing actin, ranged from 25 to 30. In the absence of a particular viral RNA, there was no signal for its probe, demonstrating the specificity of the reaction. To compare the performance of monoplex and multiplex real-time assays, 20 sera each from dengue cases and chikungunya cases were tested by both assays. There was 100 % agreement between the two assays. Clinical samples of CHIKV and DENV that had been tested earlier by conventional RT-PCR were used in the DENV/CHIKV assay to determine the sensitivity and specificity. Out of 22 CHIKV samples that were positive by the RT-PCR assay, 21 gave positive signals in the DENV/ CHIKV assay. For DENV, 51 samples that were positive by RT-PCR assay (15 DENV-1, 17 DENV-2, 14 DENV-3 and 5 DENV-4) were positive by the DENV/CHIKV assay. Twenty-one samples that were negative for DENV/CHIKV RNA by RT-PCR were negative in the real-time assay. Therefore, the test showed 100 % specificity for DENV and CHIKV, whilst the sensitivity was 95.5 % for CHIKV and 100 % for DENV. Normal human serum samples spiked with other flaviviruses (107 PFU of Japanese encephalitis virus or 106 PFU of West Nile virus) or alphaviruses (107 PFU of Semliki Forest virus and Sindbis virus) were negative in the test. In addition, three samples positive for leptospira, two for Kyasanur Forest disease virus (genus Flavivirus) and two for Crimean Congo hemorrhagic fever virus (genus Nairovirus) were found to be negative by the DENV/CHIKV assay. To determine the lower limit of detection, tenfold serial dilutions of DENV-1, 2, 3, 4 and CHIKV stocks were tested in the assay. The RNA was extracted from each dilution and tested in the DENV/CHIKV assay in duplicate. The Ct values for duplicates differed by 0 to 0.5. Linear dose response curves were obtained with regression coefficient values ranging from r2 = 0.95 to 0.99. The lower limit of sensitivity was 100 PFU/ml for DENV-2 and 3, 1000 PFU/ml for DENV-1 and CHIKV and 3250 PFU/ ml for DENV-4 (Fig. 1).
Fig. 1 Lower limit of detection. Tenfold dilutions of virus stocks of DENV-1, 2 3, 4 and CHIKV were tested in duplicate in the DENV/ CHIKV assay. The PFU/ml is plotted against the mean Ct value
To determine the applicability of the assay, serum samples from 234 febrile cases that were negative for the presence of DENV/CHIKV-specific IgM were tested. DENV RNA could be detected in 33 samples, and CHIKV RNA could be detected in 68 samples, and in one sample, CHIKV and DENV RNA was detected, suggesting a coinfection. The 33 samples containing DENV RNA were confirmed by the conventional multiplex RT-PCR, and the serotype was determined. Four samples were DENV-1 (genotype III), 13 were DENV-2 (genotype IV), 13 were DENV-3 (genotype III), and three were DENV-4 (genotype I). This also proved that the assay could detect different genotypes of DENV, as the genotype of the prototype viruses was different from the currently circulating viruses. Of the 68 CHIKV-positive samples, 20 were confirmed by conventional RT-PCR. Two genotypes of CHIKV, Asian and ECSA, were detectable. The RT-PCR-derived amplicons of two representative samples for each serotype of DENV and CHIKV were sequenced for confirmation of the result. For quantitation of viral RNA, the RNA standards prepared for DENV-1/2/3, DENV-4 and CHIKV as described above were tested at tenfold dilutions, representing 102 to 108 copies of RNA. Linear curves were obtained with R2 (regression coefficient) values of 0.99 for DENV-1/2/3, 1.0 for DENV-4 and 0.99 for CHIKV (R2 values calculated using 7500 software version 2.0.1), as shown in Figure 2. The viral load was determined for 43 clinical samples. The geometric mean ± SD value of RNA copy number (log10)/ ml in serum samples from infected patients was 6.29 ± 0.72 for DENV-1 (n = 10), 6.37 ± 0.67 for DENV-2 (n = 12), 6.60 ± 0.85 for DENV-3 (n = 9), 8.59 ± 0.05 for DENV-4 (n = 2) and 7.62 ± 0.78 for CHIK (n = 10).
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D. Cecilia et al. Fig. 2 Standard curves for viral RNA. Tenfold dilutions of the in vitro-transcribed RNA for DENV-1/2/3, DENV-4 and CHIKV were tested in duplicate in the DENV/CHIKV assay. The RNA copy number is plotted against the mean Ct value
In the present study, we report a one-step multiplex real-time RT-PCR that is capable of detecting and quantitating different genotypes of DENV serotypes and CHIKV. The DENV/CHIKV assay was a combination of the real time RT-PCR assays reported previously by our group for DENV [8] and CHIKV [12]. The sensitivity of the test for detection of DENV-4 RNA was improved by including a primer/probe set from another study [11]. Using a conventional RT-PCR assay as the standard, the sensitivity and specificity was 100 % for detection of all four serotypes of DENV. The sensitivity was lower for CHIKV (95.5 %), but the specificity was 100 %. The sensitivity for CHIKV could have been lower because of the use of stored samples. The only alternative to the conventional RT-PCR assay as the gold standard is virus isolation. Earlier reports have shown that the efficiency of RT-PCR and virus isolation for detection of dengue virus are comparable [14, 15]. The only other study reporting a multiplex real-time assay for detection of DENV/CHIKV also used conventional RT-PCR as the gold standard. They reported a lower specificity of 92.59 % [7]. The new test was able to detect DENV and CHIKV in febrile cases before the appearance of IgM, demonstrating its utility for early differential diagnosis. To conclude, we have developed a multiplex DENV/ CHIK real-time assay that can detect DENV (all four serotypes) and CHIKV with high specificity and sensitivity and can be used as an early differential diagnostic test in regions where the two viruses co-circulate. Acknowledgments We would like to thank the Director, National Institute of Virology, Pune, for his support, and Dr. Pragya Yadav for testing the KFD and CCHF samples.
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References 1. WHO (2014) http://www.who.int/mediacentre/factsheets 2. Myers RM, Carey DE (1967) Concurrent isolation from patient of two arboviruses, Chikungunya and dengue type 2. Science 157:1307–1308 3. Leroy EM, Nkoghe D, Ollomo B, Nze-Nkogue C, Becquart P, Gerard G, Pourrut X, Charrel R, Moureau G, Ndjoyi-Mbiguino A, De-Lamballerie X (2009) Concurrent chikungunya and dengue virus infections during simultaneous outbreaks, Gabon, 2007. Emerg Infect Dis 15:591–593 4. Chahar HS, Bharaj P, Dar L, Guleria R, Kabra SK, Broor S (2009) Co-infections with chikungunya virus and dengue virus in Delhi, India. Emerg Infect Dis 5:1077–1080 5. Kosasih H, de Mast Q, Widjaja S, Sudjana P, Antonjaya U, Ma’roef C, Riswari SF, Porter KR, Burgess TH, Alisjahbana B, van der Ven A, Williams M (2013) Evidence for endemic chikungunya virus infections in Bandung, Indonesia. PLoS Negl Trop Dis 7(10):e2483 6. Dash PK, Parida MM, Santosh SR, Saxena P, Srivastava A, Neeraja M, Lakshmi V, Rao PV (2008) Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection. Diagn Microbiol Infect Dis 62:52–57 7. Pongsiri P, Praianantathavorn K, Theamboonlers A, Payungporn S, Poovorawan Y (2012) Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus. Asian Pac J Trop Med 5(5):342–346 8. Gurukumar KR, Priyadarshini D, Patil JA, Bhagat A, Singh A, Shah PS, Cecilia D (2009) Development of real time PCR for detection and quantitation of dengue viruses. Virol J 6:10 9. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV (1992) Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30:545–551 10. Hasebe F, Parquet MC, Pandey BD, Mathenge EG, Morita K, Balasubramaniam V, Saat Z, Yusop A, Sinniah M, Natkunam S, Igarashi A (2002) Combined detection and genotyping of Chikungunya virus by a specific reverse transcription-polymerase chain reaction. J Med Virol 67:370–374
Assay for detection of dengue and chikungunya viruses 11. Houng HS, Chung-Ming Chen R, Vaughn DW, Kanesathasan N (2001) Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1–4 using conserved and serotype-specific 30 noncoding sequences. J Virol Methods 95:19–32. doi:10.1016/S0166-0934(01)00280-4 12. Parashar D, Paingankar MS, Kumar S, Gokhale MD, Sudeep AB, Shinde SB, Arankalle VA (2013) Administration of E2 and NS1 siRNAs inhibit chikungunya virus replication in vitro and protects mice infected with the virus. PLoS Negl Trop Dis 7(9):e2405 13. Mocellin S, Panelli M, Wang E, Rossi CR, Pilati P, Nitti D, Lise M, Marincola FM (2004) IL-10 stimulatory effects on human NK cells explored by gene profile analysis. Genes Immun 5:621–630
14. Velathanthiri VG, Fernando S, Malavige GN, Peelawatta M, Jayaratne SD, Aaskov J (2006) Comparison of serology, virus isolation and RT-PCR in the diagnosis of dengue viral infections in Sri Lanka. Dengue Bull 30:191 15. Klungthong C, Gibbons RV, Thaisomboonsuk B, Nisalak A, Kalayanarooj S, Thirawuth V, Nutkumhang N, Mammen MP Jr, Jarman RG (2007) Dengue virus detection using whole blood for reverse transcriptase PCR and virus isolation. J Clin Microbiol 45:2480–2485
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BRIEF REPORT
Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses D. Cecilia • M. Kakade • K. Alagarasu J. Patil • A. Salunke • D. Parashar • P. S. Shah
•
Received: 23 May 2014 / Accepted: 26 August 2014 Ó Springer-Verlag Wien 2014
Abstract Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaqueforming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate. Keywords Dengue chikungunya multiplex real time RT-PCR assay diagnosis Dengue virus (DENV; family Flaviviridae, genus Flavivirus) and chikungunya virus (CHIKV; family Togaviridae, genus Alphavirus) are transmitted by Aedes aegypti and Aedes albopictus mosquitoes and share the same geographic niche [1]. In the initial phase of infection, it is difficult to differentiate the two infections clinically. Whilst dengue can progress to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), CHIKV causes debilitating arthralgia, which may persist for months. Cocirculation and co-infections of CHIKV and DENV in India, Sri Lanka, Gabon, Cameroon, Madagascar,
D. Cecilia (&) M. Kakade K. Alagarasu J. Patil A. Salunke D. Parashar P. S. Shah Dengue/Chikungunya group, National Institute of Virology, Pune 411001, Maharashtra, India e-mail: [email protected]
Malaysia, Indonesia, Singapore, and Thailand have been recorded [2–5]. Differential diagnosis in these circumstances would help greatly in disease management. IgM against these viruses persists for months, and leftover IgM from a previous infection may lead to the wrong conclusion; therefore, serological methods have limitations [1]. A duplex conventional RT-PCR assay for distinguishing DENV and CHIKV [6] and a multiplex real-time RT-PCR have been reported earlier [7]. We had previously reported a two-step real-time RT-PCR for detection and quantification of DENV in human samples [8]. In the present study, we modified the assay to a one-step multiplex realtime RT-PCR assay for simultaneous detection of DENV and CHIKV. The National Institute of Virology (NIV) is a WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research and Rapid Diagnosis of Viral Diseases and routinely receives samples for diagnosis. Clinical samples that were left over after outbreak investigations were used in the study. This study was approved by the Human Ethics Committee of NIV. Waiver of the informed consent was granted by the committee on the basis of ‘‘Use of leftover specimens after clinical investigation’’ under the Indian Council of Medical Research Guidelines 2006. Prototype viruses were obtained from the virus repository of NIV. Stocks of DENV-1 (16007-genotype II, Thailand), DENV-2 (803347- genotype V, India), DENV-3 (633798 genotype I -USA), and DENV-4 (642069-genotype I, Thailand) were made in C6/36 (Aedes albopictus) cells, and CHIKV (062737- east/central/ south African (ECSA) genotype, India) in VERO (African green monkey kidney) cells. PS (porcine kidney stable) cells were used for virus titration by plaque-forming-unit (PFU) assay. PS and VERO cells were maintained in Dulbecco’s modified
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D. Cecilia et al. Table 1 List of primers and probes Primer/probe
Sequence
Genome position (accession number)
DENV-1,2,3 forward primer
GAR AGA CCA GAG ATC CTG CTG TCT
DENV1- 10647-10670 (KJ438296.1) DENV2- 10635-10658 (JX073928.1) DENV3- 10619-10642 (KJ160505.1)
DENV-1,2,3 reverse primer
ACC ATT CCA TTT TCT GGC GTT
DENV1- 10714-10694 (AY277666.2) DENV2- 10702-10682 (JX073928.1) DENV3- 10686-10666 (KF824903.1)
DENV-1,2,3 probe
AGC ATC ATT CCA GGC AC
DENV1- 10675-10691 (AY277666.2) DENV2- 10654-10670 (KJ189311.1) DENV3- 10647-10663 (KF824902.1)
DENV-4 forward primer
AAG CCA GGA GGA AGC TGT ACT CCT
10452-10475 (JQ513345.1)
DENV-4 reverse primer DENV-4 probe
CAA TCC ATC TTG CGG CGC TCT CTG TCT CTG CAA CAT CAA TCC AGG CA
10603-10583 (JQ513344.1) 10538-10563 (KF955510.1)
CHIKV forward primer
CGA AAA RGA RCC GGA GRA A
8401-8419 (JN558836.1)
CHIKV reverse primer
GAT AGT ACC CRG GKC TCA TGA CGT T
8465-8441 (JN558835.1)
CHIKV probe
CCC TRC GCA TGC TTG A
8421-8436 (JN558836.1)
Beta-actin forward primer
GGC ACC CAG CAC AAT GAA G
1044-1062 (NM_001101.3)
Beta-actin reverse primer
GCC GAT CCA CAC GGA GTA CT
1110-1091 (NM_001101.3)
Beta-actin probe
TCA AGA TCA TTG CTC CTC CTG AGA GCG C
1064-1086 (NM_001101.3)
Eagle medium (GIBCO BRL), and C6/36 in L15 (HIMEDIA). Medium was supplemented with 5 % foetal bovine serum and antibiotics. PS and VERO cultures were maintained at 37 °C and C6/36 cells at 28 °C with 5 % CO2. The viral RNA from clinical samples or cell culture fluids was extracted using a viral RNA purification kit (QIAmp Viral RNA Mini Kit, QIAGEN). Conventional RT-PCR for detection of CHIKV and serotyping of DENV were carried out as described earlier [9, 10]. The one-step multiplex real-time RT-PCR assay for DENV/CHIKV (DENV/CHIKV assay) was developed using primers and probes for DENV-1/2/3, DENV-4, CHIKV and beta-actin, which have been reported earlier [8, 11–13]. The primers and probes were targeted to regions specific to the different viruses but conserved among the genotypes. The sequences of primers and probes, along with GenBank accession numbers of the most similar sequence determined by BLAST analysis, are given in Table 1. The assay used TaqMan chemistry with minor groove binder (MGB) probes. The TaqMan probes were labeled with FAM (6carboxyfluorescein) for DENV-1/2/3, Cy5 (cyanine 5) for DENV-4, VIC (4,7,20 -trichloro-70 -phenyl-6-carboxyfluorescein) for CHIKV, and NED for beta-actin at the 50 end and BHQ-1/2 (Black Hole Quencher), a non-fluorescent quencher at the 30 end. The internal control reaction for beta-actin was carried out in a separate tube. All primers and probes were diluted to 10 pmoles/ll. The reaction mix included 0.875 ll of nuclease-free water, 5 ll
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of RNA, 12.5 ll of 2x RT-PCR buffer, 1 ll of 25X RTPCR enzyme (AgPath-ID one-step RT-PCR Kit; Applied Biosystems), 0.5 ll of three forward primers, 0.75 ll of three reverse primers and 0.625 ll of three probes specific for DENV-1/2/3, DENV-4 and CHIKV. The reaction was carried out using an ABI 7500 Real Time PCR system. The standard cycling conditions were used, which included an initial reverse transcription at 45 °C for 10 min, inactivation of reverse transcriptase at 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and annealing at 60 °C for 1 min. The detector was set to acquire emissions through FAM, Cy5 and VIC channels for each well. Threshold values were set above the background signal for each probe. On the basis of the nonspecific signal observed in non-template controls, a cutoff of cycle threshold (Ct) of 32 was decided. Samples that gave a Ct value of [32 were considered negative. For the preparation of viral RNA standards, RNA was extracted from virus stocks of DENV-3, DENV-4 and CHIKV, and the target region was amplified by RT-PCR and cloned into the TEasy cloning vector (Promega). The presence and orientation of the insert were confirmed by sequencing using the M13 primer. The plasmid was linearized by digestion with ApaI, and the target sequence was amplified using an in vitro RNA transcription kit (Roche Diagnostics). The in vitro-transcribed RNA was treated with DNase to digest the plasmid, and RNA was purified using a QIAamp Viral RNA Purification Kit (QIAGEN). The concentration of RNA was determined by spectrophotometry. The copy number of the RNA was calculated
Assay for detection of dengue and chikungunya viruses
based on the concentration of RNA and the molecular weight of each amplified target. The amplicons for DENV1, 2 and 3 were of similar length, and therefore the standard RNA of DENV-3 was used to calculate the viral load for all three viruses. Each of the primer/probe sets was tested in a monoplex real-time RT-PCR assay with RNA extracted from the prototype virus stocks containing 104 to 105 PFU. The Ct values ranged from 10 to 14. The primers and probes were then tested in the DENV/CHIKV assay using normal human serum spiked with 103 to 105 PFU of DENV-1, 2, 3, 4 or CHIKV. A positive signal was detected for FAM, representing DENV-1 (Ct = 18), DENV-2 (Ct = 14.2), DENV-3 (Ct = 18.2), for Cy5, representing DENV-4 (Ct = 21), and VIC, representing CHIKV (Ct = 12.6). The Ct values for NED, representing actin, ranged from 25 to 30. In the absence of a particular viral RNA, there was no signal for its probe, demonstrating the specificity of the reaction. To compare the performance of monoplex and multiplex real-time assays, 20 sera each from dengue cases and chikungunya cases were tested by both assays. There was 100 % agreement between the two assays. Clinical samples of CHIKV and DENV that had been tested earlier by conventional RT-PCR were used in the DENV/CHIKV assay to determine the sensitivity and specificity. Out of 22 CHIKV samples that were positive by the RT-PCR assay, 21 gave positive signals in the DENV/ CHIKV assay. For DENV, 51 samples that were positive by RT-PCR assay (15 DENV-1, 17 DENV-2, 14 DENV-3 and 5 DENV-4) were positive by the DENV/CHIKV assay. Twenty-one samples that were negative for DENV/CHIKV RNA by RT-PCR were negative in the real-time assay. Therefore, the test showed 100 % specificity for DENV and CHIKV, whilst the sensitivity was 95.5 % for CHIKV and 100 % for DENV. Normal human serum samples spiked with other flaviviruses (107 PFU of Japanese encephalitis virus or 106 PFU of West Nile virus) or alphaviruses (107 PFU of Semliki Forest virus and Sindbis virus) were negative in the test. In addition, three samples positive for leptospira, two for Kyasanur Forest disease virus (genus Flavivirus) and two for Crimean Congo hemorrhagic fever virus (genus Nairovirus) were found to be negative by the DENV/CHIKV assay. To determine the lower limit of detection, tenfold serial dilutions of DENV-1, 2, 3, 4 and CHIKV stocks were tested in the assay. The RNA was extracted from each dilution and tested in the DENV/CHIKV assay in duplicate. The Ct values for duplicates differed by 0 to 0.5. Linear dose response curves were obtained with regression coefficient values ranging from r2 = 0.95 to 0.99. The lower limit of sensitivity was 100 PFU/ml for DENV-2 and 3, 1000 PFU/ml for DENV-1 and CHIKV and 3250 PFU/ ml for DENV-4 (Fig. 1).
Fig. 1 Lower limit of detection. Tenfold dilutions of virus stocks of DENV-1, 2 3, 4 and CHIKV were tested in duplicate in the DENV/ CHIKV assay. The PFU/ml is plotted against the mean Ct value
To determine the applicability of the assay, serum samples from 234 febrile cases that were negative for the presence of DENV/CHIKV-specific IgM were tested. DENV RNA could be detected in 33 samples, and CHIKV RNA could be detected in 68 samples, and in one sample, CHIKV and DENV RNA was detected, suggesting a coinfection. The 33 samples containing DENV RNA were confirmed by the conventional multiplex RT-PCR, and the serotype was determined. Four samples were DENV-1 (genotype III), 13 were DENV-2 (genotype IV), 13 were DENV-3 (genotype III), and three were DENV-4 (genotype I). This also proved that the assay could detect different genotypes of DENV, as the genotype of the prototype viruses was different from the currently circulating viruses. Of the 68 CHIKV-positive samples, 20 were confirmed by conventional RT-PCR. Two genotypes of CHIKV, Asian and ECSA, were detectable. The RT-PCR-derived amplicons of two representative samples for each serotype of DENV and CHIKV were sequenced for confirmation of the result. For quantitation of viral RNA, the RNA standards prepared for DENV-1/2/3, DENV-4 and CHIKV as described above were tested at tenfold dilutions, representing 102 to 108 copies of RNA. Linear curves were obtained with R2 (regression coefficient) values of 0.99 for DENV-1/2/3, 1.0 for DENV-4 and 0.99 for CHIKV (R2 values calculated using 7500 software version 2.0.1), as shown in Figure 2. The viral load was determined for 43 clinical samples. The geometric mean ± SD value of RNA copy number (log10)/ ml in serum samples from infected patients was 6.29 ± 0.72 for DENV-1 (n = 10), 6.37 ± 0.67 for DENV-2 (n = 12), 6.60 ± 0.85 for DENV-3 (n = 9), 8.59 ± 0.05 for DENV-4 (n = 2) and 7.62 ± 0.78 for CHIK (n = 10).
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D. Cecilia et al. Fig. 2 Standard curves for viral RNA. Tenfold dilutions of the in vitro-transcribed RNA for DENV-1/2/3, DENV-4 and CHIKV were tested in duplicate in the DENV/CHIKV assay. The RNA copy number is plotted against the mean Ct value
In the present study, we report a one-step multiplex real-time RT-PCR that is capable of detecting and quantitating different genotypes of DENV serotypes and CHIKV. The DENV/CHIKV assay was a combination of the real time RT-PCR assays reported previously by our group for DENV [8] and CHIKV [12]. The sensitivity of the test for detection of DENV-4 RNA was improved by including a primer/probe set from another study [11]. Using a conventional RT-PCR assay as the standard, the sensitivity and specificity was 100 % for detection of all four serotypes of DENV. The sensitivity was lower for CHIKV (95.5 %), but the specificity was 100 %. The sensitivity for CHIKV could have been lower because of the use of stored samples. The only alternative to the conventional RT-PCR assay as the gold standard is virus isolation. Earlier reports have shown that the efficiency of RT-PCR and virus isolation for detection of dengue virus are comparable [14, 15]. The only other study reporting a multiplex real-time assay for detection of DENV/CHIKV also used conventional RT-PCR as the gold standard. They reported a lower specificity of 92.59 % [7]. The new test was able to detect DENV and CHIKV in febrile cases before the appearance of IgM, demonstrating its utility for early differential diagnosis. To conclude, we have developed a multiplex DENV/ CHIK real-time assay that can detect DENV (all four serotypes) and CHIKV with high specificity and sensitivity and can be used as an early differential diagnostic test in regions where the two viruses co-circulate. Acknowledgments We would like to thank the Director, National Institute of Virology, Pune, for his support, and Dr. Pragya Yadav for testing the KFD and CCHF samples.
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