Development And Application Of Real-time PCR Assay For HBV, HCV And HIV-1 Nucleic Acids

Real time PCR technology has been widely used in molecular biology and medical research due to its improved rapidity, accuracy, sensitivity, reproducibility and reduced risk of carry-over contamination. The aim of this study is to develop specific, sensitive and reproducible real-time quantitative assayss to screen for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency viruses type 1 (HIV-1) in plasma or serum samples. A multiplex real-time quantitative reverse transcription (RT-PCR) assay was also established for simultaneous detection, identification and quantification of these three viruses.Several processes for quantitative PCR have been proposed. In this study, the Taqman PCR was compared to a novel LUX (Light Upon extension) primer-based real time PCR. The results showed that the LUX PCR was not suit to screen for viruses because it allowed two-step RT-PCR only and provided a poor reproducibility. Real-time TaqMan PCR was a more specific, sensitive and reproducible alternative for quantitation of both viral DNA and RNA levels of HBV, HCV and HIV-1.Virus DNA or RNA preparation from serum (or plasma) is a key step for real time PCR assay, as it affects the accuracy of quantitation of viral load directly. Here we reported an effective and rapid nucleic acid extraction system based on magnetic beads. Nucleic acids were bound to the beads in the presence of high concentrate of chaotropic salt (Guanidine thiocyanate) and were eluted at a slightly alkaline pH. The recovery efficiency is>80% for DNA and>70% for RNA. We compared the detection sensitivity of the new method with the sensitivities of the QIAamp Viral DNA Mini Kit and QIAamp Viral RNA Mini Kit and the results showed a good correlation. The characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.Three Taqman-based real time PCR assays were developed and evaluated in this study, which were designed to provide specific, reproducible and highly sensitive method for viral load determination of HBV, HCV and HIV-1. The assays employed an effective and rapid nucleic acid extraction system based on magnetic beads. The performance of the real time assays were validated by testing serial dilutions of HBV DNA, HCV RNA, and HIV-1 RNA (10 to 106 copies per reaction), respectively, and a good linear relationship was obtained between the Ct values and the log10 concentration of the DNA or RNA. The assays possessed high sensitivity and the detection limits of these systems were as few as 50 copies/ml of serum. These assays provide ideal tools for monitoring the treatment efficacy and studying the relationship between viral load and stage of disease.A multiplex real-time quantitative reverse transcription (RT-PCR) assay was also established for simultaneous detection and quantification of HBV, HCV and HIV-1 in plasma or serum samples. The preferred dyes of FAM, HEX and Cy5 and a preferred primer-probe combination of HBV, HCV and HIV-1 were chosen for triplex assays. The results indicated that genomic amplification of one virus was unaffected by the simultaneous amplification of the other two. The efficacy of the multiplex assay was evaluated by testing serial dilutions of HBV DNA, HCV RNA, and HIV-1 RNA (10 to 106 copies per reaction) in one tube. Three linear standard curves were obtained between 101 and 10 copies/reaction. The detection limits of this system were as few as 50 copies/ml for HBV DNA, HCV RNA and HIV-1 RNA. The multiplex PCR was compared to the singleplex PCR and showed a good correlation. This assay has the potential to be used for large-scale nucleic acid testing (NAT) of blood donations.The HBV real time PCR assay was employed to explore the relationship between the serum contents of HBV DNA and the HBV serological markers. Serum samples whose HBsAg and HBV DNA had been determined to be positive were assayed again by Elisa for HBeAg, anti-HBc anti-HBs and anti-HBe. Ten modes of HBV M were found among the 121 HBsAg and HBV DNA positive persons. Higher copy levels of serum HBV DNA occurred mainly in patients with HBeAg positive mode. However, some HBeAg negative carriers have high levels of HBV DNA. AFP was also detected by Elisa and 18 samples were determined to be primary liver cancer (AFP> 400 ng/ml). All these samples were HBeAg positive and 12 samples contain low levels of HBV DNA.In conclusion, it is significant to combine the HBV serological markers with HBV DNA level, which can better reflect the real conditions of HBV infection and replication in the patients with HBV.