The Clinical Study Of Hepatitis B Viral P Gene Mutations Detected By MALDI-TOF MS And Direct DNA Sequencing

OBJECTIVE:To compare the differences between two methods of MALDI-TOF MS and PCR-direct DNA sequencing on hepatitis B viral P gene mutations detection.METHODS:Study group consisted of 90 serum samples from 90 chronic hepatitis B patients received nucleoside analogues (NA) therapy for more than 1 year and HBV-DNA titer still higher than 500copies/ml; 10 serum samples from 10 chronic hepatitis B patients never treated with antiviral therapy and HBV-DNA titer higher than 1×105copies/mL comprised control group. The 90 samples included in study group then divided into 6 groups according to the different NA therapy. Next, the HBV P gene mutations were detected by MALDI-TOF MS and PCR-direct DNA sequencing at the same time, using TYPE4.0 software and Sequence Navigator software to analysis the two methods results separately.RESULTS:1. In study group, mutations were detected in 53 samples and total mutation sites were 86 by MALDI-TOF MS, however, by PCR-direct DNA sequencing, just 19 samples were found mutations and total 29 mutation sites were detected, the mutations detection rate of MALDI-TOF MS was higher than PCR-direct DNA sequencing and the difference was statistical significance (P<0.05). In control group, mutations were not detected whether by MALDI-TOF MS or by PCR-direct DNA sequencing.2. For study group, in 12 samples with HBV-DNA titer between 500-1000 copies/ml,6 samples were detected mutations by MALDI-TOF MS, the detection rare was 50%. On the contrary, no mutation was detected by PCR-direct DNA sequencing. Similarly, at the other comparable lever of HBV-DNA titer, the mutations detection rate of MALDI-TOFMS also was higher than PCR-direct DNA sequencing.This result difference still was statistical significance (P<0.05)3. Among the mutations detected samples by MALDI-TOF MS, sera from patients prescripted with Lamivudin or Telbivudine, the detected mutation sites were completely corresponded with the clinical known mutation sites special for Lamivudin, the consistent rate was 100%; however,sera from patients prescriped with Adefovir, the detected mutation sites were just partly corresponded with the clinical known mutation sites special for Adefovir, the consistent rate was 70%.CONCLUSIONS:1. The mutations detection rate of MALDI-TOF MS was higher than PCR-direct DNA sequencing.2. MALDI-TOF MS had higher sensitivity for known mutation sites detection compared with PCR-direct DNA sequencing. 3. MALDI-TOF MS had high consistent rate and was a accurate method for mutation detection.4. MALDI-TOF MS was a rapid, sensitive and accurate method and could be used for monitoring mutations in chronic hepatitis B patients treated with NA therapy.