Simultaneous detection of HBV and HCV by multiplex PCR normalization

Abstract

AIM:To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV. METHODS:Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method. RESULTS:The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%, respectively for both HBsAg and anti-HCV positive patients, and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165% and 83.3%, respectively for anti-HCV positive patients. CONCLUSION:The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.Wang N, Gao XQ, Han JX. Simultaneous detection of HBV and HCV by multiplex PCR normalization.

Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus

“…8 Wang et al demonstrated that the sensitivity of multiplex normalized PCR method was 78.6%, 75%, and 83.3% for the detection of HBV DNA, HCV RNA, and super-infection of HBV and HCV, with specificity of 80%, 90%, and 70%, respectively. 9 The superinfection rate of HBV and HCV was very high and is a leading cause of chronic hepatitis and hepatocellular carcinoma. [20][21][22] Konomi et al 23 reported a multiplex PCR method for simultaneous detection of hepatitis B, C, and G viral genomes, where the levels of concordance with the data obtained by conventional single PCR method were 100% for single infection, 98-100% for double infection, and 92% for triple infection.…”

“…Thus, the multiplex PCR becomes a highly cost-effective method because of the reduction in labor and reagent and faster detection. [8][9][10] Multiplex PCR has been used for detection of HBV and coinfection with both HAV and HCV. 11 The present study was designed with the aim to develop a rapid, reliable, and cost-effective multiplex PCR assay for the simultaneous detection of both DNA and RNA containing hepatitis viruses which are highly prevalent in India such as HBV, HCV, and HEV.…”

Hepatitis C Virus Prevalence in Haemodialysis Patients From Three Centers in Baghdad, Iraq: A Survey By Polymerase Chain Reaction and Serological Methods

“…In contrast, none of the anti-HCV negative patients were shown to be viremic by the PCR as mention by De Albuquergue et al (2005). The detection of HCV RNA in non-hepatitis patients could be explained by the fact that the patients might be in the early stage of acute hepatitis, and the antibody had not been produced yet (Wang et al, 2004). On the other hand, one cannot exclude the existence of impaired immune responses in some of these patients (Schneeberger et al, 1998).…”

“…Possible explanations for this low percentage of viremic patients in our population include: the level of viremia may be low and below detectable level of PCR assay at time of sampling, the pattern of fluctuating viremia or intermittent viremia, patients might be cured from HCV infection at time of sampling (Bdour, 2002;Al-Kubaisy et al, 2003;and Wang et al, 2004). PCR-based methods reliability may further be compromised if viral RNA is lost in the serum or plasma through storage or improper laboratory handling or if it is absent from the circulation during sample collection (Zein, 2000).…”

Diagnosis of hepatitis C virus infection by enzyme‐linked immunosorbent assay and reverse transcriptase‐nested polymerase chain reaction: A comparative evaluation

“…When the virus was cleaned up, only the antibody was positive, the nucleic acids could not be detected. Hence, the detection rate of PCR was lower when ELISA was used as a golden standard (24).…”