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The Journal of Molecular Diagnostics, Vol. -, No. -, - 2015

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Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy Q30

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Lida Chen,*y Wenli Li,z Kuo Zhang,y Rui Zhang,y Tian Lu,*y Mingju Hao,*y Tingting Jia,*y Yu Sun,y Guigao Lin,y Lunan Wang,y and Jinming Li*y From the National Center for Clinical Laboratories,* Beijing Hospital, Beijing; the Graduate School,y Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing; and the China-Japan Friendship Hospital,z Beijing, People’s Republic of China Accepted for publication July 24, 2015.

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Address correspondence to Jinming Li, National Center for Clinical Laboratories, Beijing Hospital, 1 Dahua Rd., Dongdan, Beijing, People’s Republic of China. E-mail: jmli@nccl. org.cn.

Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a real-time quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kitsdCobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The real-time quantitative RT-PCR method delivered a good performance, with a linearity of R2 Z 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R2 Z 0.97), with a means  SD value of 0.06  1.96 log IU/mL (0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This real-time quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. (J Mol Diagn 2015, -: 1e8; http://dx.doi.org/10.1016/ j.jmoldx.2015.07.009)

Viral infections cause acute or chronic diseases, even cancer, in humans worldwide.1 Viruses that commonly infect humans include both DNA and RNA viruses.2e4 Current standard-ofcare therapy for most DNA or RNA virus infections is antiviral treatment.5 Quantitative nucleic acid testing has become a gold standard marker for clinical decisions regarding the use of antiviral therapy.6 The most recently developed nucleic acid testing method is the real-time PCR-based assay.7 However, commercially available assays require strict conditions for sample collection, transportation, storage, and processing because of the instability of viral nucleic acids,8 in particular viral RNAs.9 The instability of viral nucleic acids is due to decay mechanisms. For example, the 50 -untranslated region (50 -UTR) of hepatitis C virus (HCV) is the most conserved region.10 Thus, it is used for design targets in many commercial HCV

RNA detection kits. However, the 50 -UTR has unique decay mechanisms due to its secondary structure. The 50 -UTR includes a region of the HCV internal ribosome entry site element,11 which can be hybridized with the liver-specific microRNA (miR-122), to unlock and switch the 50 -UTR to an open structure,12 and the RNA secondary structure can also melt with increasing temperatures to an open conformation.13 The opened conformation induces the exposure of the 50 end of the 50 -UTR to exonuclease-mediated degradation.14 The HCV 50 -UTR strand can fold into several stemeloop secondary structures,15 and the loop structures are more sensitive Supported by grant 2013ZX10004805 (program 863) from the National High Technology Research and Development Program of China. L.C. and W.L. contributed equally to this work. Disclosures: None declared.

Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2015.07.009

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125 to cleavage by endonuclease than are the stem structures.16 As 126 Q9 described above, the 50 -UTR of HCV RNA is prone to 127 cleavage into short pieces of different lengths.14,16 128 Thus, when RNA is cleaved during sample collection, 129 handling, and storage, decreased detection sensitivity or 130 even a false-negative result may be obtained using the 131 currently available commercial kits. For example, when 132 serum samples from HCV-infected patients were stored at 133 70 C for up to 9 years, a significant loss of HCV RNA 134 135 load was detected using Cobas Amplicor Monitor assay17 136 (Roche Diagnostics Deutschland GmbH, Mannheim, Ger137 Q10 many), and a 23% decrease in HCV RNA was observed in 138 samples stored at 20 C for 6 months when measured using 139 Q11 the branched DNA assay.18 140 To overcome the drawbacks of these commercial kits and to 141 minimize the impact of the instability to nucleic acid testing, we 142 developed a real-time quantitative RT-PCR (RT-qPCR) 143 method based on a new primer design strategy. We tested our 144 method and new strategy with HCV RNA as a model. 145 146 147 Materials and Methods 148 149 Primer/Probe Design Strategy 150 151 Due to the decay mechanisms of HCV 50 -UTR described 152 Q12 above, primers were designed according to the following 153 rules: i) the 50 -UTRs of HCV genotypes 1 to 7 were aligned 154 to identify conserved regions, which were selected as 155 candidate regions for primer/probe design; ii) the 50 -UTR 156 contains many stemeloop structures (Figure 1A), and we ½F1 157 chose primer/probe sets from conserved sequences on stem 158 structures, trying to avoid the loops structures; iii) because 159 160 the base-pairing structures of the 50 -UTR might open at 161 increasing temperatures or by miR-122 hybridization, the 162 sequences near the 50 end of the 50 -UTR are susceptible to 163 cleavage by 50 -30 exonucleases (Figure 1B), and therefore 164 the probe and primers were designed close to the 30 end of 165 the 50 -UTR to prevent exonuclease degradation; iv) over166 lapping regions among the forward primer, probe, and 167 reverse primer were avoided; and v) the covering loops were 168 as few as possible for the target amplified fragment, which 169 means that shorter amplified fragments were better. 170 171 172 Serum Sample Collection 173 174 We randomly collected 81 blood samples and their genotyping 175 data from HCV-infected patients from the Beijing Ditan Hos176 pital (Beijing, China) from May 2014 to October 2014. The 177 blood samples were centrifuged immediately for the collection 178 of serum. Each serum sample was divided into two aliquots. 179 One aliquot was directly stored at 80 C as the HCV RNA 180 nondegraded sample for baseline. The other aliquot was divided 181 into two parts. For one part, 81 samples were incubated at room 182 183 temperature for 6 hours and then stored at 80 C as the HCV 184 RNA degraded sample. For the other part, 42 samples were 185 randomly selected from the 81 collected samples and incubated 186

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Figure 1

Primer/probe sets design strategyebased mechanisms. A: Nuclease cleavage site and primer/probe sets of 5’ untranslated region (UTR). Circles indicate endonuclease cleavage sites. B: Schematic illustration of secondary structure unlocked by miR-122 hybridizing or temperature increasing. Down arrow shows the first site where exonuclease-mediated degradation starts. Yellow symbol shows the 50 -30 exonuclease at present place. Pink symbol show the 50 -30 exonuclease at past place. DT, temperature increase; F0 , complementary to forward primer F; miR-122, liver-specific mRNA; P0 , complementary to probe P; R1, R2, R3, and R4, reverse primers for 62-, 157-, 222-, and 304-Bp targets, respectively.

at 37 C for 6 hours and then stored at 80 C as HCV RNA further-degraded samples. These treatment conditions simulated the maximum time delay between sample collection and detection observed in some Chinese hospitals in developing areas, at room temperature, and during hot summers. All aliquots were thawed at room temperature when ready for analysis. The study protocol was approved by the Ethics Committee of the National Center for Clinical Laboratories, and the study was conducted in accordance with the guidelines of the Declaration of Helsinki.

RNA Isolation Two protocols were used for serum RNA purification. The QIAamp-based method was used for analysis of the HCV RNA isolates using QIAamp Viral RNA Mini Kit (catalog no. 52904; Qiagen, Germantown, MD) according to the manufacturer’s instructions. The miRelute-based method was used for HCV RNA isolates using the QIAamp-based method, but the QIAamp column was substituted with a

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A Primer Design Strategy for HCV PCR Table 1

Sets of Primers and Probe

Primer/probe F P PCR target R1 PCR target R2 PCR target R3 PCR target R4

Function

Sequence

Location

Forward primer* Hydrolysis probe*

5 -TGCACGGTCTACGAGAC-3 FAM-50 -CCGGGGCACTCGCAAGCACCC-30 -GMB

322e339y 398e319y

Reverse primer

50 -GCCTTGTGGTACTGCCTGAT-30

277e297

Reverse primer

50 -CGACCGGGTCCTTTCTTGGAT-30

182e203

Reverse primer

50 -GACCCCCCCTCCCGGGAGAG-30

117e137

Reverse primer

50 -ATCACTCCCCTGTGAGGAACT-30

35e56

0

0

size 62 Bp size 157 Bp size 222 Bp size 304 Bp

*The forward primer and hydrolysis probe were matched with reverse primers R1, R2, R3, and R4, respectively. y Locations of forward and probe means the position of their complementary sequences.

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miRelute column from a MiRcute Kit (Beijing Tianjin Bioengineering Corporation, Beijing, China), which contained a special silicon membrane to isolate short RNA fragments [as small as 200 nt).

Real-Time RT-qPCR for HCV RNA Detection Reactions of 100 mL contained 20 mL of PCR buffer (Qiagen); 4 mL of dNTP Mix (Qiagen); 20 mL of OneStep RT-PCR Enzyme Mix (Kehua Bio-engineering Co., Ltd., Shanghai, China); 2 mL of 10-mmol/L forward primer F (InvitroGen, Carlsbad, CA); 2 mL of 10-mmol/L reverse primer R (InvitroGen); 2 mL of 10-mmol/L hydrolysis minor groovebinding probe (InvitroGen), which was labeled with the fluorophore carboxyfluorescein at the 50 end and quencher minor groove-binding at the 30 end; and 50 mL of template RNA. Thermal cycling was performed on an Applied Biosystems 7500 instrument [AB Sciex, Foster City, CA (formerly Applied Biosystems)] under the following conditions: 50 C, 25 minutes; 94 C, 2 minutes; 5 cycles of 94 C for 10 seconds, 55 C for 15 seconds, and 72 C for 15 seconds; and 42 cycles of 94 C for 10 seconds, 60 C for 45 seconds, and 40 C for 30 seconds. Fluorescence was measured at 60 C for each cycle.

Performance Evaluation Our new real-time RT-qPCR method included miRelutebased RNA isolation and real-time RT-qPCR with the most optimized primer/probe set (F, P, and R1) targeting a 62-Bp ½T1 fragment (Figure 1A and Table 1). National standard material for HCV RNA (GBW09151; 2.26eþ2, 2.26eþ3, 3.97eþ4, and 8.5eþ5 IU/mL) was used for detecting the HCV RNA concentration in the clinical sera. The detection performance was evaluated as follows.

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Limit of Detection The limit of detection (LOD) of our method was determined by the analysis of the second HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques [National Institute for Biological Standards and Control (NIBSC) code 08/264], including genotypes 1a, 1b, 2, 3, 4, 5, and 6. Each genotype

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was diluted using an HCV-negative serum sample from a healthy blood donor to the following concentrations: 103, 500, 100, 50, 25, 12.5, and 6.25 IU/mL. Five replicates per concentration were tested and then analyzed by probit regression. Precision The intra-assay variation was assessed by testing four clinical samples with different viral loads (106, 105, 103, and 102 IU/mL) 10 times in a single run, whereas the interassay variation was assessed by testing the same samples 10 times in 10 separate runs. Linearity Linearity of the duplex real-time RT-qPCR method was determined using serial 10-fold dilutions of a clinical sample diluted with an HCV-negative serum to the following concentrations: 6.49eþ7, 6.49eþ6, 6.49eþ5, 6.49eþ4, 6.49eþ3, and 6.49eþ2 IU/mL. At each concentration, three replicates were tested in a single run. Specificity The specificity of the new real-time RT-qPCR method was determined by analysis of HCV genotypes 1 to 6 (NIBSC code 08/264) and 102 HCV RNAenegative serum samples from blood donors and patients infected with different pathogens (viral, bacterial, and yeast), including hepatitis A virus, hepatitis B virus, HIV, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Candida albicans. Agreement with Commercial Kit The performance of the new real-time RT-qPCR method was compared to that of Cobas AmpliPrep/Cobas TaqMan Q18 (CAP/CTM; Roche) by analyzing nondegraded serum Q19 specimens from 81 HCV-infected patients. The operation steps of the CAP/CTM kit were performed according to the instructions provided by the manufacturer.

Different Target Lengths and Position for 50 -UTR Amplification To verify the effects of the target length and position on the RNA-amplification rate, we designed three additional

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Chen et al

Figure 2 Performance evaluation of our method of real-time quantitative RT-PCR (RTqPCR). A: Linearity of the duplex real-time RT-qPCR assay, linear relationship between the quantification cycle (Cq) values, and the log hepatitis C virus (HCV) RNA concentration is Y Z 3.51x þ 41.06 (R2 Z 0.99); the blank Ct value (no template control) is undetectable. B: Correlation of our realtime RT-qPCR method and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay in detecting 81 nondegraded sera, the Pearson bivariate correlation coefficient (R2) is 0.97 (P < 0.0001).

reverse primers from genotype-conserved sequences to compare with that of R1. The three reverse primers (R2, R3, and R4) were designed for amplifying 157-, 222-, and 304-Bp PCR targets, respectively (Figure 1 and Table 1). Forty-two baseline samples and their corresponding further-degraded serum samples treated at 37 C were analyzed in this test. Agarose gel electrophoresis was used for detecting primer dimers and nontarget hybridization.

Clinical Diagnostic Performance The clinical performance of the new real-time RT-qPCR method was compared to that of the CAP/CTM and the Kehua HCV commercial kits. The CAP/CTM kit is a widely used assay worldwide, and the Kehua kit is a HCV RNA detection kit developed in and commonly used in China. Eighty-one nondegraded serum samples and their corresponding degraded samples (treated at room temperature) were analyzed using the three assays. Additionally, 42 nondegraded samples and their corresponding further-degraded samples (treated at 37 C) were tested. The CAP/CTM and the Kehua assays were performed according to the manufacturers’ instructions.

numbers AB691953.1, AB690461.1, AB792683.1, AB795432.1, KC844046.1, DQ480524.1, and EF108306.2) Q20 and alignments were generated using ClustalX 2.1 (http://www. clustal.org/clustal2, last accessed July 11, 2015). Secondary structure of the HCV 50 -UTR was predicted by MFOLD (http:// mfold.rna.albany.edu/?qZmfold, last accessed July 11, 2015). Primer Premier version 6.0 (Premier Software Inc., Cherry Hill, NJ) was used for predicting the Tm and hairpin of the designed primers and probe using our new strategy. Statistical analyses were performed with the IBM SPSS Statistics version 19.0 Q21 (IBM Corp., Armonk, NY) and GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA). The Bland-Altman plots were used for the analysis of agreement between the assays. Probit regression was performed to determine the LOD. Differences between groups were assessed using the U-test. Correlation and linear regression were calculated by Pearson t-test with 95% CI. The significance level was set at P < 0.05.

Results New Isolation Method Is More Efficient in Detecting HCV RNA

Statistical Analysis Sequences of HCV genotypes 1 to 7 were searched from GenBank (http://www.ncbi.nlm.nih.gov/genbank; accession Table 2

We compared the QIAamp-based method and our miRelute-based method by isolating 42 HCV RNA furtherdegraded serum samples, and the RNAs were amplified by

LOD of HCV Genotype by the Described real-time RT-qPCR Method Hit ratey in second HCV RNA genotype panel (NIBSC code: 08/264)

HCV RNA input,* IU/mL

1a

1b

2

3

4

5

6

All

1000 500 100 50 25 12.5 6.25 LOD

5/5 5/5 5/5 5/5 3/5 3/5 1/5 42.3

5/5 5/5 5/5 5/5 4/5 3/5 0 29.4

5/5 5/5 5/5 5/5 4/5 2/5 0 29.9

5/5 5/5 5/5 5/5 3/5 1/5 0 36.4

5/5 5/5 5/5 5/5 4/5 2/5 1/5 33.6

5/5 5/5 5/5 4/5 4/5 3/5 1/5 42.5

5/5 5/5 5/5 4/5 3/5 1/5 0 59.2

35/35 35/35 35/35 33/35 25/35 15/35 3/35 42.6

*HCV RNA input was detected by Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay. y Hit Rate means positive results per parallel tests performed. HCV, hepatitis C virus; LOD, limit of detection; NIBSC, National Institute for Biological Standards and Control; RT-qPCR, quantitative RT-PCR.

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A Primer Design Strategy for HCV PCR

real-time RT-qPCR with a primer/probe set targeting a 62-Bp fragment. In our study, the viral load of HCV RNA isolated by miRelute-based method was significantly greater than that isolated by the QIAamp-based method (P < 0.05). Because the real-time RT-qPCR was designed to target a 62-Bp fragment, the miRelute-based method showed better performance in isolating shorter RNAs than did the QIAamp-based method.

HCV RNA Detection Method Presents a Good Evaluation Performance Using the reverse primers R1, R2, R3, and R4, the viral load was 62 Bp > 157 Bp > 222 Bp > 304 Bp in furtherdegraded samples (P < 0.05, P < 0.01, and P < 0.001, respectively), only the 62-Bp primer set maintained a similar load distribution in nondegraded and corresponding further-degraded samples (Supplemental Figure S1). Thus, the new real-time RT-qPCR method was optimized by miRelute-based RNA isolation and real-time RT-qPCR with the primer/probe set targeting the 62-Bp fragment. The evaluation of our new real-time RT-qPCR method indicated high-quality performance.

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559 560 561 562 563 564 565 566 Figure 3 Clinical performance of our method compared to those of commercial kits. Bland567 Altman plot analysis for 81 hepatitis C virus 568 (HCV) samples. The samples with the means and 569 1.96 SDs are indicated. The heavy solid line in570 dicates the mean differences (log IU/mL). The 571 dashed line indicates the means  1.96 SD (log 572 IU/mL). HCV RNA viral load deviation between the 573 real-time quantitative RT-PCR (RT-qPCR) method 574 and Kehua (A and B) and between the real-time 575 RT-qPCR method and Cobas AmpliPrep/Cobas 576 TaqMan (CAP/CTM) (C and D) assay in nondegraded serum samples (n Z 81) (A and C) and 577 degraded samples treated at room temperature 578 for 6 hours (n Z 81) (B and D). E: Deviations of 579 clinical genotypes 1b (n Z 46), 2a (n Z 26), 580 and 6a (n Z 9) between nondegraded and 581 degraded sera detected by our method (log 582 stable RNA e log unstable RNA), each box shows Q28 583 the median (interquartile range) (box length, 584 containing 50% of data), and whiskers show 585 extreme values. No significant differences are 586 shown among genotypes 1b, 2a, and 6a. 587 588 589 590 591 592 593 594 Linearity 595 596 Linear relationship between the Ct values and the log HCV 597 RNA concentration yielded Ct Z 3.51x þ 41.06 and 2 R Z 0.99 (Figure 2A). ½F2 598 599 600 LOD 601 The LOD of our method was determined for genotype panel 602 1a, 1b, 2, 3, 4, 5, and 6. The original quantifications of the 603 panel tested using our new real-time RT-qPCR method were 604 eþ3 eþ4 eþ4 eþ4 eþ3 eþ5 6.99 , 3.01 , 1.92 , 1.35 , 5.23 , 1.29 , and 605 eþ4 5.27 IU/mL, respectively. The probit regression indi606 cated high sensitivity across all genotypes (Table 2). The ½T2 607 overall LOD was as low as 42.6 IU/mL (95% CI, 32.84 to 608 67.76 IU/mL). The conversion factor of our method was 609 calculated to be 1 IU/mL Z 2.4 copies/mL. 610 611 Precision 612 The intra-assay CV ranged from 1.24% to 2.33% (SD, 0.29 613 614 to 0.72), and the interassay CV ranged from 1.06% to 3.34% 615 (SD, 0.35 to 0.91). 616 617 Specificity 618 HCV genotypes 1 to 6 were positive. A total of 102 blood 619 donors and the infected patients with other pathogens (virus, 620

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Chen et al 621 622 623 624 625 626 627 628 629 630 631 632 633 Figure 4 Clinical performance of the described method in further-degraded sera. The dashed lines indicate the original viral load before degradation. A: 634 Hepatitis C virus (HCV) RNA viral load in nondegraded serum samples (n Z 42), detected by the described real-time quantitative RT-PCR (RT-qPCR) method (62 635 Bp) and the Kehua and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) kits. There are no significant differences between the results obtained using the real-time 636 RT-qPCR method and those obtained using Kehua or CAP/CTM. B: HCV RNA viral load in degraded serum samples treated at room temperature for 6 hours 637 (n Z 42), detected by the real-time RT-qPCR method and the Kehua and CAP/CTM kits; the viral load detected using the real-time RT-qPCR method is significantly greater than those detected using the Kehua and CAP/CTM kits. C: HCV RNA viral loads in further-degraded sera treated at 37 C for 6 hours 638 (n Z 42), detected by the real-time RT-qPCR method and the Kehua and CAP/CTM kits; the real-time RT-qPCR method yields viral loads significantly 639 greater than those of the Kehua and CAP/CTM kits. *P < 0.05, **P < 0.01, and ***P < 0.001. 640 641 642 643 bacteria, and yeast) showed negative results for HCV RNA Additionally, the efficiency of our method was greater than 644 amplification. The specificity of the new real-time RT-qPCR that of the commercial kits in detecting degraded sera. The 645 method was 100%. genotypes of the 81 patients were 1b (n Z 46), 2a 646 (n Z 26), and 6a (n Z 9), respectively. The deviations 647 Agreement with Commercial Kits of genotypes 1b, 2a, and 6a in nondegraded and 648 The results obtained using the new real-time RT-qPCR degraded samples were not significantly different, sug649 method were highly concordant with those obtained using the gesting that genotype has no effect on HCV viral load 650 CAP/CTM assay in detecting 81 nondegraded sera. The cordetection with our new method (Figure 3E). On agarose 651 relation coefficient (R2) was 0.97 (P < 0.001) (Figure 2B). gel electrophoresis, the length of the targets detected 652 The Bland-Altman plot indicated that the results obtained using the CAP/CTM and Kehua kits ranged from about 653 using our method were 0.06 log IU/mL (1.96 SD, 0.38 to 200 to 300 Bp, and our four designed primer/probe sets 654 655 Q22 0.25 log IU/mL) less than those obtained using the CAP/CTM produced bands at the correct positions without primer 656 assay; none of the samples showed differences of >0.5 log dimers and nontarget hybridization, also confirmed by 657 ½F3 IU/mL (Figure 3C). sequencing. 658 To determine whether our method maintains a good 659 performance in further-degraded HCV RNA detection, 42 Clinical Performance Is Superior to That of Commercial 660 serum samples treated at 37 C for 6 hours were tested Kits in HCV RNAeDegraded Serum Samples 661 using our method, and the results were compared to those 662 obtained using the CAP/CTM and Kehua kits. The results The clinical performance of our method was compared to 663 obtained using our method were not significantly different that of the CAP/CTM and Kehua kits. Eighty-one non664 from those obtained using the CAP/CTM or Kehua kit in degraded and corresponding degraded sera treated at room 665 nondegraded samples (Figure 4A). However, in the cor- ½F4 temperature were detected. The Bland-Altman plots with 1.96 666 667 SD of agreement are shown in Figure 3. The results obtained responding degraded samples (room temperature), the 668 using our method were in high agreement with those obtained viral loads detected using the CAP/CTM and Kehua kits 669 with the Kehua kit (0.04  0.29 log IU/mL) (Figure 3A) and were decreased, and performance with our method was 670 the CAP/CTM kit (0.06  0.31 log IU/mL) (Figure 3C) significantly greater than that of the Kehua kit (P < 0.01) 671 before degradation, but they were significantly different from and the CAP/CTM kit (P < 0.05) (Figure 4B). In the 672 those obtained with the Kehua and CAP/CTM kits after further-degraded samples treated at 37 C (Figure 4C), the 673 degradation; the mean value was 0.66 log IU/mL greater than viral loads detected using the CAP/CTM and Kehua kits 674 that obtained with the Kehua kit, with a larger range of 1.96 were decreased dramatically compared with the results 675 SD (0.81 to 2.14 log IU/mL) (Figure 3B) and 0.23 log obtained using nondegraded (Figure 4A) and degraded 676 IU/mL greater than that obtained with the CAP/CTM kit, (Figure 4B) serum, especially when using the Kehua kit. 677 with a greater range of 1.96 SD (0.71 to 1.17 log IU/mL) Viral loads yielded using our method were significantly 678 679 (Figure 3D). In summary, the results obtained with our greater than those detected using the CAP/CTM kit 680 method were in agreement with those obtained using (P < 0.05) and the Kehua kit (P < 0.001). Ours was the 681 commercial kits in detecting nondegraded sera. only method that yielded a detection rate that was similar 682

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A Primer Design Strategy for HCV PCR among nondegraded, degraded, and further-degraded samples (Figure 4).

Discussion

Q23

Viruses may cause serious human diseases.19 The accurate determination of viral load is important for selecting antiviral therapy.20 However, viral nucleic acids, especially viral RNAs, are unstable, which may lead to a decline in nucleic acid concentration when using the currently available commercial kits. To overcome the impact of instability on viral load detection, we developed a new primer design strategy using HCV RNA as a model. Based on the HCV RNA 50 -UTR secondary structure and cleavage mechanism, a suitable primer/probe set targeting a 62-Bp fragment was developed using this strategy. We found that if the targeted template is short and devoid of nuclease cleavage sites, the RNA detection will be more sensitive even if samples are exposed to unstable conditions. HCV RNA isolation is an important step. The QIAampbased method is commonly used for RNA isolation,21 but it is not suitable for isolating RNA fragments of shorter than 200 nt, according to the manufacturer’s instructions. Therefore, we improved the extraction method using a miRelute columneadsorbing RNA even shorter than 100 nt. The concentration of RNA purified by our miRelute-based method was significantly greater than that obtained using the QIAamp-based method, suggesting that HCV RNA has been degraded into small fragments and that the miRelutebased method is more capable of adsorbing shorter RNA fragments to increase the sensitivity in detecting degraded HCV RNA samples. To verify the detection performance of our real-time RTqPCR method, a series of evaluation indicators were assessed. The linear regression analysis was good. Our method also had good sensitivity in detecting genotypes 1 to 6, with a total LOD of 42.6 IU/mL. According to the manufacturer’s instructions, the LOD of the Kehua kit was 500 IU/mL and that of the CAP/CTM kit was 15 IU/mL, suggesting that our method has lesser sensitivity compared with that of the CAP/CTM kit and greater sensitivity compared with the Kehua kit. The lesser sensitivity of our method may be related to the sample volume required in the RNA isolation step (CAP/CTM, 1000 mL; our method, 140 mL). In addition, our real-time RT-qPCR method presented high specificity and good precision. Moreover, correlation analysis and the Bland-Altman plot suggested that the results obtained using our method were in good agreement with those from commercial kits. To support the correctness of the primer design strategy, we also designed three gradually lengthened primersdR2, R3, and R4dtargeting 157-, 222-, and 304-Bp fragments, respectively (Figure 1A and Table 1) and compared their performance with the R1. Figure 1 shows that the detection

The Journal of Molecular Diagnostics

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rate increases as the length of the PCR target decreases if RNA samples are unstable. The most likely explanation of this finding is that, in sera and in the environment, the endonuclease may cleave inside the loop structures and/or exonuclease may cleave from the 50 end of the 50 -UTR.15,16 Based on our strategy, the 62-Bp target was located on the stem structure with rare cleavage sites (Figure 1A) and located close to the 30 end of the 50 -UTR to avoid 50 exonucleaseemediated decay14 when an RNA secondary structure opens (Figure 1B). In contrast, as the targets get longer, the templates span more loop structures and get closer to the 50 end of the 50 -UTR, causing a gradual increase in the degradation frequency. Because our method performed well in the methodology evaluation, we then assessed whether it is suitable for detection in clinical samples. The CAP/CTM assay kit is widely used for HCV viral load testing worldwide,23 and the Q24 Kehua kit is an HCV RNA detection kit developed in and widely used in China.24 In the Bland-Altman plots presented in Figure 3, the performance of our method compares favorably to that of commercial HCV assays with nondegraded samples and is superior to those of the commercial assays with degraded samples. The findings in Figure 4 suggest that when HCV RNA was further degraded, the detection efficiency of commercial kits decreased continuously. In contrast, detection in degraded and furtherdegraded sera was not significantly decreased when using our real-time RT-qPCR method. These findings can be explained by our new primer/probe design strategyebased mechanisms. Although the primer sequences used in the CAP/CTM and Kehua kits are proprietary, their target lengths range from approximately 200 to 300 Bp by agarose gel electrophoresis. Because the 50 -UTR of HCV is only 341 nt,25 CAP/CTM and Kehua PCR targets will inevitably span many loop structures and are close to the 50 end of the 50 UTR, allowing for endonuclease/exonuclease degradation. These findings suggest that our method has advantages over existing assays in a modern clinical setting. For example, to avoid decreased detection efficiency, serum samples need to be transported at 2 C to 8 C or even at

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial ...
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