Quantification and genotyping of hepatitis B virus in a single reaction based on dual molecular beacon real-time PCR

Abstract

Objective To develop a new method for simultaneous quantifying and genotyping of HBV in a single reaction based on dual molecular beacon real-time PCR. Methods Genotype B and C recombinant plasmids were constructed as the standards and genotype-specific primers and molecular beacons were designed for each genotype.The molecular beacons of genotype B and C were labeled with FAM and Hex respectively.In this way,a simultaneous qualification and genotyping method for HBV DNA in a single real-time PCR reaction system was developed.Firstly,10-fold gradient dilution of genotype B and C standard plasmids (103-1011 kIU/L) were utilized to evaluate the linear ranges and sensitivity of this approach.The clinical specificity was tested with twenty different serum specimens (5 cases with hepatitis C virus,5 cases with herpes simplex virus and 5 cases with human papilloma virus as well as 5 healthy volunteers) ; the reproducibility was assessed by intra-assay and inter-assay coefficient of variation (CV) of cycle threshold (Ct) value through 10 repeated detections within a batch and between batches of the B,C standard plasmids (108,106and 104kIU/L).Then the accuracy of qualifying and genotyping of the self-built method was evaluated by a parallel examination with 132 HBV infected patients by use of two commercial kits as the references.Finally,these HBVpositive patients were divided into 4 groups:asymptomatic carrier (n=21),chronic hepatitis (n=77),liver cirrhosis (n=25) and hepatocellular carcinoma (n=9) to investigate the relationship of genotypes,stages of disease progression and HBV DNA load. Results A simultaneous qualification and genotyping assay was successfully built and its genotyping sensitivity was 103kIU/L and the linear range was 103-1011k IU/L.The intra-assay CV of B genotyping was 1.51% to 1.80% and the interassay CV was 2.11% to 3.03%,while the intra-assay CV of C genotyping was 1.79% to 1.95% and the inter-assay CV was 2.53% to 2.91%.The results of non HBV infected cases and healthy volunteers showed negative.In the test of 132 HBV infected patients,the general coincident rate of genotyping results comparing our assay and HBV DNA genotyping kit was 90.9% (120/132,Kappa=0.832,P<0.05).The HBV DNA quatitive results between the assay[5.07 (3.89-6.33)]and HBV DNA quatitive kit[5.19 (4.15-6.32) lg kIU/L] were well correlative (R2=0.8477,P<0.05).69 genotype B cases,51 genotype C cases and 12 B/C mixed-genotype cases were detected by dual molecular beacon real-time PCR method and their HBV DNA load were 4.54 (3.83-6.17),5.53 (4.02-6.55),4.58 (3.68-4.98) lg kIU/L respectively.Where the patients with genotype C had higher DNA load than the patients with other two genotypes (Z=-2.195 and-2.162,P<0.05).The HBV DNA load of asymptomatic group,chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group were 7.02 (6.35-7.84),4.94 (4.16-6.25),4.37 (3.50-5.17) and 3.45 (3.25-4.92) lg kIU/L, respectively.Among them,the asymptomatic group was significantly higher than those of other three groups (Z=-4.244,-4.568 and-3.489,P<0.001) and DNA load comparing with the chronic hepatitis group,liver cirrhosis group and hepatocellular carcinoma group also showed statistically different (Z=-2.894 and-2.413,P<0.05).However,compared with the liver cirrhosis group and hepatocellular carcinoma group there was no significant difference (Z=-0.995,P=0.335). Conclusion A dual molecular beacon real-time PCR assay which can simultaneously quantifying and genotyping HBV DNA with highly accuracy,sensitive and specificity is successfully developed.(Chin J Lab Med,2013,36:333-338) Key words: Hepatitis B virus; DNA; viral; Genotype; Polymerase chain reaction