1. Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy 
Lida Chen, Wenli Li, Kuo Zhang, Rui Zhang, Tian Lu, Mingju Hao, Tingting Jia, Yu Sun, Guigao Lin, Lunan Wang, Jinming Li 
The Journal of Molecular Diagnostics 
Volume 18, Issue 1, Pages (January 2016)
DOI: /j.jmoldx
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Primer/probe sets design strategy–based mechanisms. A: Nuclease cleavage site and primer/probe sets of 5' untranslated region (UTR). Circles indicate endonuclease cleavage sites. Letters highlighted in green indicate the start codon for viral protein synthesis. 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 5′-3′ exonuclease at present place. Orange symbol show the 5′-3′ exonuclease at past place. ΔT, temperature increase; F′, complementary to forward primer F; miR-122, liver-specific mRNA; P′, complementary to probe P; R1, R2, R3, and R4, reverse primers for 62-, 157-, 222-, and 304-Bp targets, respectively.
The Journal of Molecular Diagnostics  , 84-91DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Performance evaluation of our method of quantitative RT-PCR (RT-qPCR). A: Linearity of the duplex RT-qPCR assay, linear relationship between the quantification cycle (Cq) values, and the log hepatitis C virus (HCV) RNA concentration is Y = −3.51x (R2 = 0.99); the blank Ct value (no template control) is undetectable. B: Correlation of our real-time 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).
The Journal of Molecular Diagnostics  , 84-91DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Clinical performance of our method compared to those of commercial kits. Bland-Altman plot analysis for 81 hepatitis C virus (HCV) samples. The samples with the means and 1.96 SDs are indicated. The heavy solid line indicates the mean differences (log IU/mL). The dashed line indicates the means ± 1.96 SD (log IU/mL). HCV RNA viral load deviation between the quantitative RT-PCR (RT-qPCR) method and Kehua (A and B) and between the RT-qPCR method and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) (C and D) assay in nondegraded serum samples (n = 81) (A and C) and degraded samples treated at room temperature for 6 hours (n = 81) (B and D). E: Deviations of clinical genotypes 1b (n = 46), 2a (n = 26), and 6a (n = 9) between nondegraded and degraded sera detected by our method (log stable RNA – log unstable RNA), each box shows the median (interquartile range) (box length, containing 50% of data), and whiskers show extreme values. No significant differences are shown among genotypes 1b, 2a, and 6a.
The Journal of Molecular Diagnostics  , 84-91DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Clinical performance of the described method in further-degraded sera. The dashed lines indicate the original viral load before degradation. A: Hepatitis C virus (HCV) RNA viral load in nondegraded serum samples (n = 42), detected by the described quantitative RT-PCR (RT-qPCR) method (62 Bp) and the Kehua and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) kits. There are no significant differences between the results obtained using the 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 (n = 42), detected by the RT-qPCR method and the Kehua and CAP/CTM kits; the viral load detected using the 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 (n = 42), detected by the RT-qPCR method and the Kehua and CAP/CTM kits; the RT-qPCR method yields viral loads significantly greater than those of the Kehua and CAP/CTM kits. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
The Journal of Molecular Diagnostics  , 84-91DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Supplemental Figure S1
The distribution of hepatitis C virus (HCV) RNA viral load detected by four primer/probe sets. A: HCV RNA viral load in nondegraded serum samples (n = 42), detected by 62-, 157-, 222-, and 304-Bp methods. There are no significant differences. B: HCV RNA viral load in degraded serum samples (n = 42), detected by 62-, 157-, 222-, and 304-Bp methods. The 62-Bp viral load is significantly greater than that of the 157-, 222-, and 304-Bp groups. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
The Journal of Molecular Diagnostics  , 84-91DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions