Study On The Detection Method Of Hepatitis C Virus Reverse Transcription Fluorescence Quantitative PCR Based On Novel Primer Design Strategy

Virus infection is harmful to human health worldwide, leading to acute or chronic diseases, even cancer and death. The viruses that infect humans are primarily DNA or RNA viruses, such as EB virus, human papilloma virus, hepatitis B virus, hepatitis C virus, Human immunodeficiency virus, etc. Prevailing treatment is antiretroviral therapy, and viral load detection is an important basis for treatment decisions, medication doses and prognosis. The gold standard for viral nucleic acid detection is quantitative nucleic acid testing (NAT), and real-time PCR detection method is commonly used. However, the viral nucleic acid of specimen is unstable under inappropriate collection, transport, storage and handling conditions. If using the current commercial kits for quantitative detection, the instability of viral nucleic acid detection will lead to lower efficiency, or even false negative resuLts. Our goal is to establish a method, which can detect the original viral load of the patient as much as possible, even when the viral nucleic acid is unstable, to overcome the effects of the instability on NAT by current commercial kits. Because HCV RNA is typically unstable, we take it as as the research object of this article.Based on the degradation mechanism of HCV RNA, we developed a new primer design strategy, which in addition to adhering to the classic primer design principles primer design for the conserved regions, also proposed that primer design shoμLd avoid the restriction sites and make the fragment length as short as possible, to reduce the risk of template degradation. Currently, commercial kits often design primers in the HCV RNA 5’non-coding region (5’-UTR), which rarely take secondary structure and restriction sites for PCR amplification into consideration. When the virus nucleic acid is unstable, the secondary structure and cleavage sites are likely to affect the efficiency of commercial kits, and get reduce viral load, or even false negative resμLts. Therefore, based on the primer design strategy, we established a new HCV RNA fluorescence quantitative RT-PCR (reverse transcription quantitative real-time PCR, RT-qPCR) method. The method is comprised by the new primers/probe, the extraction of HCV RNA and HCV RNA amplification. RNA was extracted using QIAGEN RNA method which is widely used, but the extraction column was used as MiRcute column, which can adsorbe RNA fragments even less than 100nt. RNA amplification is used herein primers and probes designed in accordance with the new primer design strategies and the new cycling conditions for RNA reverse transcription and amplification. To validate the advantages of the new extraction method (MiRcute extraction column), we compared our method with QIAGEN RNA extraction kit by simμLtaneously extraction and amplification of HCV RNA degradation samples for viral load testing. To verify the performance of the new RT-qPCR method, a methodology detection for RT-qPCR were used, including lineary, the limit of detection (LOD), sensitivity, specificity, and detection kit and commercialization of relevance. To validate its clinical utility, we carried out RT-qPCR and the CAP/CTM(?) and KeHua(?) methods to detect clinical specimens, and compared their resμLts statistically. To verify the correctness of the primer design strategy, we designed another three pairs of primers to extend the fragment, and the four pairs of viral load were statistically compared, too.The results showing that, first of all, a new extraction method (MiRcute extraction column) is better than QIAGEN RNA extraction method (p<0.037), which is capable of adsorbing more degradation HCV RNA of small fragments, thereby increasing amplification efficiency. Second, based on the new primer design strategy, the new RT-qPCR method has a good methodology detection performance. The Lineary R2=0.99; the LOD was 46.06 IU/mL (40.65-54.07 IU/mL,95% confidence interval); the intra-assay coefficient of variation in the sensitivity was 0.93% to 1.34%, inter-assay CV was 1.06% to3.34%; the specificity was 100%; with the high correlation (R=0.957) with CAP/CTM(?) commercial kits. Third, the new RT-qPCR method has good clinical utility. When the viral nucleic acid was stable, this method has no statistically significant (p> 0.05) with commercial kits CAP/CTM(?) and KeHua(?) in detecting clinical samples; however, when the viral nucleic acid is unstable, this method is statistically significant (p<0.05) from commercial kits CAP/CTM(?) and KeHua(?). When the virus nucleic acid is unstable, the viral load is decreased by CAP/CTM(?) and KeHua(?), and KeHua(?) decline more, while our new RT-qPCR method can still reach the original viral load in HCV RNA degraded serum as much as possible. Fourth, in the RNA degradation sample, with the extension of the target fragment, RNA amplification efficiency decreases, ie, the amplification efficiency 62-bp> 157-bp> 222-bp> 304-bp. The shorter fragment and less restriction sites, the smaller chance of being digested. The results prove the correctness of the new primer design principles.For developing the primer design strategy, we creatively take secondary structure and the nuclease sites into account, and successfully established a new method of RT-qPCR basing on the new primer design strategy. The method overcomes effect of the instability of viral nucleic acid on commercial kits due to specimen collection, transport, storage and handling. Using our RT-qPCR method, unstable viral nucleic acid can still be detected and the viral load can reach to the original value as much as possible, which is important for clinical HCV diagnosis, treatment and prognosis. Meanwhile, due to the secondary structure and cleavage sites of the viral nucleic acid, a new primer design strategy can also be applied to the detection of other RNA or DNA of the virus, and laid the foundation for the development of new testing methods for other viruses.