Detection of Hepatitis B Virus M204V Mutation Quantitatively via Real-time PCR

Abstract

Background and Aims: Drug-resistant DNA mutations of the hepatitis B virus (HBV) affect treatment response in chronic hepatitis B patients. We have established a new, sensitive, specific, accurate and convenient real-time PCR method to detect HBV mutations quantitatively. Methods: Blood samples were collected from patients showing viral breakthrough, primary nonresponse, or poor response during treatment, and mutations were detected via direct sequencing to assess our method. A plasmid containing the M204V mutation was synthesized and standard curves plotted. Results: The determination coefficient for linear correlation between Ct and log plasmid copy numbers was 0.996, where Ct value was −3.723log (DNA concentration) +48.647. Coefficients of variation indicated good reproducibility. Correctness was within tolerable bias. Limit of detection was 10 3 copies/ mL. Specificity, accuracy, positive predictive value and negative predictive value were 92.86%, 100%, 96.88%, 100% and 94.74%, respectively. Conclusions: These results show that our method can be used to detect HBV M204V mutations with the advantages of sensitivity, specificity and efficiency, providing a new choice for monitoring drug resistance.

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

“…The real-time PCR technique has also recently been applied to quantitatively detect hepatitis B virus (HBV) M204V mutation [ 29 ]. This is an amino acid substitution in the hepatitis B viral polymerase linked to viral resistance to nucleotide analogs, the main treatment option for patients suffering from chronic hepatitis B.…”

“…For quantitative measurement, a plasmid carrying the M204V mutation was synthesized. The method showed advantages in terms of sensitivity, specificity, and efficiency in detecting HBV M204V mutations and provided a new option for monitoring drug resistance [ 29 ]. A mismatch amplification mutation assay for rapid detection of Neisseria gonorrhoeae , the causative agent of the sexually transmitted infection gonorrhea, has been developed using a real-time PCR platform.…”

A Simple and Rapid Method for Quantitative Detection of Hepatitis B Virus Drug-resistant Mutations

“…According to the results, Liang et al 17 confirmed probes 204-2-VP2 and 204-2-IP2 with a positive rate of 100% and the false positive rate of 0%. Furthermore, the amplification curves showed a highly linear relationship between Ct values and the amount of serially diluted plasmid DNA for the primers and probes (R 2 =0.996, slope=−3.723), indicating an appropriate quantitative detection of M204V/I.…”

“…To counter the disadvantages of the methods above, Liang et al 17 developed a real-time PCR assay aimed at monitoring the reaction process and quantitatively detecting HBV resistance mutations. Compared with the above-mentioned commonly used methods, this method has a streamlined operation process and can detect resistance mutations with a rate of less than 10%, whereas its disadvantage is that it can only detect known mutation sites by using designed probes and primers.…”

“…Finally, a linear standard curve for quantification was obtained by use of a 10-fold dilution of the plasmid, and the formula was used to calculate the viral load within extracted DNA. 17 Real-time PCR can quantify the mutated gene with relatively accurate measurement in units of copies/mL, while the current method uses ab approximate description via the percentage of mutation.…”