Multiplex ligation-dependent probe real-time fluorescence PCR (Polymerase Chain Reaction) kit for detecting drug resistance of HBV (Hepatitis B Virus) lamivudine and/or adefovir

Abstract

The invention belongs to the biotechnology field and particularly relates to a multiplex ligation-dependent probe real-time fluorescence PCR (Polymerase Chain Reaction) technology for detecting the drug resistance of chronic hepatitis B. A multiplex ligation-dependent probe real-time fluorescence PCR kit comprises an MLPA (Multiplex Ligation-dependent Probe Amplification) probe set, a filling sequence and a universal primer, wherein probes are respectively designed according to mutation sites rtM204V, rtM204I, rtA181T, rtA181V and rtN236T of a P gene reverse transcription region of HBV (Hepatitis B Virus); the sequences of the probes are represented by SEQ ID NO: 1-10. The invention further relates to a method for detecting the drug resistance of the HBV lamivudine and adefovir by virtue of the kit. The method combines the high throughput of an MPLA technology, and the rapidness, sensitivity and real-time detection of real-time fluorescence PCR, and has a high coincidence rate compared with a direct sequencing method, and can be used for detecting samples which cannot be detected by the sequencing method due to insufficient sensitivity; a whole detection process takes 4-5 hours; the clinic hepatitis B drug resistance detection method provided by the invention is low in cost and quick in detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a real-time fluorescent PCR technology of multiple connection probes for detecting drug resistance of chronic hepatitis B. Background technique [0002] Chronic hepatitis B remains a major public health problem worldwide. In China, two drugs, lamivudine and adefovir, have become the first-line drugs for the treatment of chronic hepatitis B. However, the generation of HBV drug resistance has become the main obstacle to the success of long-term treatment of chronic hepatitis B. Therefore, it is very important to detect whether the HBV infected by patients is resistant to lamivudine and adefovir for the selection of clinical anti-HBV drugs. At present, a variety of methods for the detection of hepatitis B drug resistance have been reported, including direct sequencing of PCR products, reverse linear hybridization (INNO LIPA), gene chip and real-time PCR and other technolog...

AI Technical Summary

Problems solved by technology

However, the traditional MLPA technology is used, that is, the use of capillary electrophoresis to quantitatively analyze the DNA target sequence to be tested or the use of chip hybridization technology to quantify and type the pathogenic nucleic acid, which requires the synthesis of a large number of probes of different lengths, or the need for probes Complex operation steps such as fixation, hybridization and elution

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