Study Of Primer-specific Fluorescent Real-time PCR On The Detection Of Spontaneous Mutation Of HBV YMDD Motif

Chronic hepatitis B virus (HBV) infection severely threaten public health. Antiviral therapy plays an important role by inhibiting the replication of the virus. More antiviral nucleoside analogues have been approved for the antiviral therapy against HBV. However, mutation in the YMDD motif of the polymerase gene frequently occurs with Lamivudine or Entecavir after long-term antiviral therapy which bring on drug-resistance. At present, it has been reported that spontaneous YMDD mutation existed in patient with CHB who have never received any antiviral treatment, which challenge the application of nucleoside analogues in patients with CHB.In this study, we first constructed those Plasmids of YMDD,YVDD,YIDD with molecular methods,and then evaluated three assays for detection of YMDD mutation. We detected spontaneous YMDD mutation in 196 untreated subjects with primer-specific real-time PCR and analyzed the factors related to natural YMDD mutation .Part I Construction 0f plasmids of YMDD,YVDD,YIDD and evaluation of three assays for detection of HBV YMDD motif mutationObjective To evaluate three assays for detection of HBV YMDD motif mutation and choose an optimal assay for next detection .Methods Plasmids were constructed and tested with molecular methods. Three assays, including direct sequencing of PCR products, probe-specific and primer-specific fluorescent real-time PCR, were assessed respectively,based on mixed plasmids of different proportion, samples from some patients with CHB who have never received any antiviral treatment and from 105 CHB patients who were resistant to LAM.Results Plasmids of YMDD,YVDD,YIDD were constructed successfully. In mixed plasmids of different proportion, some certain plasmids would not be detected by direct sequencing if it accounted for less than 25% of the total plasmids. The test of the sensitivity of probe-specific fluorescent real-time PCR show that it will be liable to bring false negativity or imprecision and be not fit for the detection of the spontaneous mutation of HBV YMDD motif. Primer-specific fluorescent real-time PCR were tested in high sensitivity and specificity.Certain mutants would be detected by this assay if it accounted for less than 10% of the total viruses.In 105 CHB patients resistant to Lamivudine, certain mutants were detected by direct sequencing and primer-specific fluorescent real-time PCR in 97 patients and 95 patients,respectively. In 110 untreated subjects, we find that 11 patient was detected in their serum by primer-specific fluorescent real-time PCR,and only one patient was detected in their serum by direct sequencing.Conclusions Direct sequencing of PCR products, probe-specific fluorescent real-time PCR and primer-specific fluorescent real-time PCR are all applicable for genetic resistance detection in those patients with phenotypic resistance. Direct sequencing and probe-specific fluorescent real-time PCR are not fit for the detection of the spontaneous mutants.Primer-specific fluorescent real-time PCR is better than other two assays in the detection of the spontaneous mutants and can analyse coexisting strains quantitatively.Part II Incidence of spontaneous HBV YMDD motif mutation and its related factorsObjective To analyze the incidence of spontaneous HBV YMDD motif mutation and its related factors.Methods Serum samples collected from 196 untreated subjects with CHB were detected by primer-specific fluorescent real-time PCR. Those related factors including gender, age, HBeAg status, viral load of serum, state of illness, genotype of virus and the time of virus's infection were analysed with statistic method.Results Among 196 subjects with CHB, spontaneous mutants of YMDD were detected in 21 subjects (20 YVDD subject and 1 YIDD subject). YMDD variants account for >50%, 25%～50%, 9%～25% of total virus load in 1, 5, 15subjects, respectively. Gender, age, HBeAg status, viral load, state of illness and the time of virus's infection were not found to be significantly related with spontaneous YMDD mutation by logistic regression analysis, while genotype B were associated with higher spontaneous mutations of YMDD than genotype C.Conclusions Spontaneous YMDD mutants exist in patients with CHB, and are not related with gender, age, HBeAg status, viral load of serum, the time of virus's infection or state of illness.Higher spontaneous mutations tend to emerge in genotype B virus than in genotype C virus.