| Hepatitis B virus(HBV)is a DNA virus that endangers human health seriously.HBV DNA quantification play a critical role in the management of HBV chronic infections.At present,the common method for HBV DNA quantitation are real time PCR.There were only a pair of primer and probe in most commercial kits.However,HBV was a highly genetic variant DNA virus and drug-resistant mutations emerged with the use of anti-viral drugs.If the mutations occurred and sequence mismatched with primer/probe in the commercial kit,it would lead to the underestimation of the sample.Therefore,it is necessary to develop a new HBV quantification method to reduce the risk of underestimation of HBV DNA titre.We developed a new duplex real-time PCR assays which contained two pairs of primers and two probes based on the conserved S and C regions of HBV genome.This method includes the design and screening of primers/probes,the establishment and optimization of PCR reaction system.We validated its linearity,the limit of detection(LOD),specificity,and so on to verify the performance of the new quantitative method,and compared the quantitative values of the new quantitative method with the commercialization kit COBAS TaqMan HBV Test version 2 as well as the Daan Quantitative Kits to verify their clinical utility.Samples with the difference in the quantitative values of more than 10 times were amplified and sequenced,and the amplified products were inserted into T vector,which would be quantitatively verified.The results showed that the newly established duplex primer/probe HBV DNA quantification method exists high detection performance.The performance of this novel quantitative method was comparable to that of the commercialized COBAS TaqMan HBV Test version 2 as well as the Daan Quantitative Kits.We found that the quantitative values of the COBAS TaqMan HBV Test version 2 and Daan Quantitative Kits were 10 times lower than new method in some samples.The sequencing results showed that there were mutations in nt1962 and 1963 of the binding region of reverse primer of the Cobas TaqMan HBV Test version 2 reagent.Verifying with plasmids containing these mutated sequences,we demonstrated that the Cobas TaqMan HBV Test version 2 reagent had a low quantitation value.It was showed that the quantitation values of the S and C regions were comparable in the duplex primer/probe HBV DNA quantification method(R2=0.9657).But it could not avoid low quantitative value or missing detections of C region in some samples,which existed mutations in the primer binding regions.The novel duplex primer/probe HBV DNA quantification method has strong clinical practicality and reduces the risk of low quantitative value because of the mismatches between single primer/single probe and template,and provides accurate basis for clinical diagnosis,treatment and prognosis of HBV.HBV nucleic acid test(NAT)is a significant component of blood nucleic acid test,and plays an important role in preventing HBV transmission and ensuring blood transfusion safety.HBV is a highly variant DNA virus.There were a risk of HBV DNA false-negative result using NAT reagents with mono-target in HBV genome.In this study,we have established a HBV nucleic acid test method based on the conserve region in S and C regions.We validated its the limit of detection(LOD),specificity,and so on to verify the performance of the new qualitative method.The single positive samples detected by Haoyuan and Cobas Taq Screen MPX Test,version 2 NAT reagents were detected by the method.Then the HBV DNA quantitation were further performed.The results showed that the newly established method exists good detection performance.Among the single positive samples detected by Haoyuan and Cobas Taq Screen MPX Test,version 2,the HBV DNA concentration of 8 samples were higher than the LOD of NAT reagents.This implies there is a higher risk of fale-negative detection by using mono-target HBV DNA test and dual-target mehod could reduce the risk. |
