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Application of primer probe combination and kit thereof in HBV detection

A technology of primer probes and kits, applied in the biological field, can solve the problems of drug resistance mutations, false negatives, missed detection of HBV infection, etc., and achieve the effects of high detection efficiency, strong specificity, and good sensitivity

Pending Publication Date: 2019-06-07
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a combination of primers and probes and the application of its kit in HBV detection, which is used to solve the problem of missed detection of HBV infection in the prior art due to the low content of hepatitis B virus in the body and prone to base mutations. Negative occurrence, drug-resistant mutations, occult HBV infection, etc.

Method used

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  • Application of primer probe combination and kit thereof in HBV detection
  • Application of primer probe combination and kit thereof in HBV detection
  • Application of primer probe combination and kit thereof in HBV detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Single-plex S gene region fluorescent quantitative PCR HBV quantitative detection

[0148] 1. Dissolve the standard positive template with distilled water and dilute it to 5×10 5 copies / μL, 5×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL, 5×10 1 copies / μL, 5×10 0 copies / μL.

[0149] 2. Fluorescent quantitative PCR experiment (10 μL amplification reaction system): 5.0 μL fluorescent quantitative reaction solution, 0.35 μL (0.5 μM) forward primer of the S gene region primer set, 0.35 μL (0.5 μM) reverse primer of the S gene region primer set , fluorescent probe 0.75 μL (0.2 μM), primer probe set selected from combination S1, standard positive template 3.5 μL.

[0150] Fluorescent quantitative PCR reaction parameters are as follows: pre-denaturation: temperature is 95°C, time is 10min; denaturation: temperature is 95°C, time is 10s, annealing: temperature is 62°C, time is 30s, 45 cycles of denaturation and annealing, this step consists of The Light Cycler 480Ⅱ ...

Embodiment 2

[0152] Quantitative detection of HBV by fluorescence quantitative PCR of single C gene region

[0153] Fluorescent quantitative PCR experiment (10 μL amplification reaction system): 5.0 μL fluorescent quantitative reaction solution, 0.35 μL (0.5 μM) forward primer of C gene region primer set, 0.35 μL (0.5 μM) reverse primer of C gene region primer set, fluorescent Probe 0.75 μL (0.2 μM), primer probe set selected from combination C1, standard positive template 3.5 μL.

[0154] Fluorescent quantitative PCR reaction parameters are as in Example 1, and the results are shown in the attached figure 2 .

Embodiment 3

[0156] Quantitative detection of HBV by fluorescence quantitative PCR of single X gene region

[0157] Fluorescent quantitative PCR experiment (10 μL amplification reaction system): 5.0 μL fluorescent quantitative reaction solution, 0.35 μL (0.5 μM) forward primer of the X gene region primer set, 0.35 μL (0.5 μM) reverse primer of the X gene region primer set, fluorescent Probe 0.75 μL (0.2 μM), primer probe set selected from combination X1, standard positive template 3.5 μL.

[0158]Fluorescent quantitative PCR reaction parameters are as in Example 1, and the results are shown in the attached image 3 .

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Abstract

The invention provides application of a primer probe combination and a kit thereof in HBV detection. The primer probe combination is at least one selected from the group consisting of an S gene regionprimer probe combination, a C gene region primer probe combination, and an X gene region primer probe. Primer probes are designed in conserved sequences of S, C and X genomes of hepatitis B virus, and uses fluorescent quantitative PCR technique to simultaneously detect HBV-DNA in a same tube; and the primer probe combination is simple and rapid, and improves the sensitivity and specificity of thedetection method of hepatitis B virus, minimizes the chance of false negative results due to mutations, and further improves detection efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a primer-probe combination and its kit in HBV detection. Background technique [0002] Hepatitis B virus (HBV) infection is one of the most serious public health problems in the world. The total number of people infected with HBV in the world used to be as high as 2 billion. Since the discovery of hepatitis B virus infection, it has been receiving great attention, especially in recent years, the potential clinical harm of chronic hepatitis B virus infection and low viral load occult hepatitis B virus infection (Occult HBV infection, OBI) has become more and more serious. Attention has greatly increased the risk of liver cirrhosis, primary liver cancer, liver failure, and HBV transmission. About 1 million people die of diseases related to HBV infection every year. Although a highly effective hepatitis B vaccine has been available since 1982, there are still more th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12N15/11C12Q1/706C12Q2600/16C12Q1/6818
Inventor 赵耀杨宇婷
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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