Simultaneous detection of HBV and HCV by multiplex PCR normalization.
OBJECTIVE
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.


CONCLUSIONS
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.
Shandong Medicinal Biotechnological Center, Shandong Academy of Medical Sciences, Key Laboratory for Biotechdrugs, Ministry of Public Health, Jinan 250062, Shandong Province, China.
Ning
N
Wang
Xue-Qin
XQ
Gao
Jin-Xiang
JX
Han
D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006509:Hepatitis B / Q000150:complications / Q000175:diagnosis*; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000150:complications / Q000175:diagnosis*; D018937:Hepatitis C Antibodies / Q000097:blood; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D006514:Hepatitis B Surface Antigens; D018937:Hepatitis C Antibodies; D011933:Reagent Kits, Diagnostic; D004247:DNA