Abstract
Quantum dots (QDs) are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV) gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB) patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 103 IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation.
Highlights
78 million people are chronic hepatitis B virus surface antigen (HBsAg) carriers with serological evidence, of which about 20–30 million are chronic hepatitis B (CHB) patients inChina [1,2]
Primers were designed for the mutant gene of hepatitis B virus (HBV) polymerase M204I, which were confirmed by the serum samples containing M204I mutation previously detected by sequencing
The results show that HBV DNA polymerase gene mutation was detected by the newly The results show that polymerase mutation was detected by the4), newly established quantum dot-labeled probe togene detect gene mutations
Summary
78 million people are chronic hepatitis B virus surface antigen (HBsAg) carriers with serological evidence, of which about 20–30 million are chronic hepatitis B (CHB) patients in. The INNO-LiPA (INNO: Innogenetics N.V., Ghent, Belgium; LiPA: an assay based on the line probe assay) method [13] shows higher sensitivity than direct sequencing, and appears to be a rapid assay for the detection of HBV genotypic resistance. It is not suitable for clinical promotion due to the high costs involved. We developed a QDs-mediated fluorescence method for detecting the hepatitis B virus gene mutation with features including low cost, high throughput, high sensitivity and accuracy
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