Application of PCR‐LDR‐nucleic acid detection strip in detection of YMDD mutation in hepatitis B patients treated with lamivudine

Abstract

Chronic hepatitis B virus (CHBV) infection causes cirrhosis and hepatocellular carcinoma. Lamivudine (LAM) has been successfully used to treat CHBV infections but prolonged use leads to the emergence of drug-resistant variants. This is primarily linked to a mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the HBV polymerase gene at position 204. Rapid diagnosis of drug-resistant HBV is necessary for a prompt treatment response. Common diagnostic methods such as sequencing and restriction fragment length polymorphism (RFLP) analysis lack sensitivity and require significant processing. The aim of this study was to demonstrate the usefulness of a novel diagnostic method that combines polymerase chain reaction (PCR), ligase detection reaction (LDR) and a nucleic acid detection strip (NADS) in detecting site-specific mutations related to HBV LAM resistance. We compared this method (PLNA) to direct sequencing and RFLP analysis in 50 clinical samples from HBV infected patients. There was 90% concordance between all three results. PLNA detected more samples containing mutant variants than both sequencing and RFLP analysis and was more sensitive in detecting mixed variant populations. Plasmid standards indicated that the sensitivity of PLNA is at or below 3,000 copies per ml and that it can detect a minor variant at 5% of the total viral population. This warrants its further development and suggests that the PLNA method could be a useful tool in detecting LAM resistance.

Monitoring Genetic Population Biomarkers for Wastewater-Based Epidemiology

“…After incubation, the amplification products were detected by the lateral flow device, following the manufacturers' instructions. 23 A positive test was indicated by the presence of two red lines: one assigned to the target sample and another corresponding to the control that confirms that the test is valid. Results ( Figure 1A) show no difference between the two heating methodology.…”

“…23 The amplicon of LAMP was transferred with a pipet to the sample pad of the lateral flow device, for detection. A positive test was indicated by the presence of two red lines, one assigned to the target sample and another corresponding to the control, which confirms if the test is valid.…”

“…19 At the end of the reaction, the amplicon produced was transferred to a lateral flow device to be read visually (Scheme 1). The working principle of the commercial lateral flow device, as previously published, 23 is illustrated in Figure S1 and detailed in the Materials and Methods. The entire assay, from the sample to the answer, is completed within ∼45 min.…”

Association between clinical features and YMDD mutations in patients with chronic hepatitis B following lamivudine therapy

“…It has previously been demonstrated in clinical studies of LAM therapy that drug resistance may appear following YMDD mutation (33). The YMDD motif is the binding site of LAM (8,9); thus, the occurrence of YMDD mutations may affect LAM binding with the YMDD motif.…”

Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism

“…In this context, for the genotypic evaluation of antiviral resistant hepatitis B virus subpopulations, various monitoring methods have been developed as alternatives to commonly used direct DNA sequencing method. These include, PCR-RFLP method [1,7], PCR-RFMP (restriction fragment mass polymorphism) method [4], methods using conventional PCR with allele specific primers [10,19,20], peptide nucleic acid (PNA) mediated PCR clamping method [8], methods based on real-time PCR [3,5,12,13,17,18] and methods based on immobilized oligonucleotide probes [6,9,11,15,21,22]. The sensitivity of these methods in detecting antiviral resistant HBV subpopulations has been reported to be 5 % in PCR-RFLP method [1], 1 % in PCR-RFMP method [4], 0.5 % in allele specific PCR method [10], 0.01 % in PNA mediated PCR clamping method [8], depending on the experimental settings 10-0.01 % in real-time PCR methods [3,5,12,13,17,18] and 30-5 % in methods using immobilized oligonucleotide probes [6,9,11,21,22].…”