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Real-time fluorescence nucleic acid isothermal amplification detection kit for HCV (hepatitis C virus)

A hepatitis C virus, constant temperature amplification detection technology, applied in the field of virus biomedical detection, can solve the problems of inability to detect 6 HCV genotypes, poor specificity and sensitivity, etc., and achieves easy automation and coverage. Wide and accurate measurement of the effect

Active Publication Date: 2016-04-13
SHANGHAI RENDU BIOTECH
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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 fast, high accuracy, easy to control pollution, simple equipment, low-cost hepatitis C virus RNA detection kit, while solving the specificity of the hepatitis C detection kit in the prior art. The sensitivity is not good, and the technical problems of not being able to detect all HCV6 genotypes

Method used

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  • Real-time fluorescence nucleic acid isothermal amplification detection kit for HCV (hepatitis C virus)
  • Real-time fluorescence nucleic acid isothermal amplification detection kit for HCV (hepatitis C virus)
  • Real-time fluorescence nucleic acid isothermal amplification detection kit for HCV (hepatitis C virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1 Design of special primers and probes for real-time fluorescent nucleic acid constant temperature amplification and detection of hepatitis C virus (HCV)

[0124] The present invention selects the nucleic acid of hepatitis C virus 5'non-coding region that has no secondary structure and is highly conserved as the amplified target sequence region (its nucleotide sequence is shown in SEQ ID NO: 1), and the primer probe is designed to obtain The specific sequence is as follows:

[0125] (1) nT7 primer and T7 primer used to amplify hepatitis C virus target polynucleotide. The nucleotide sequence of T7 primer is: 5'-AATTTAATACGACTCACTATAGGGAGAGGCCTTTCGCGACCCAACACTAC-3' (sequence 3); the nucleotide sequence of nT7 primer is: 5'-GCGTTAGTATGAGTGTCGT-3' (sequence 4);

[0126] (2) The detection probe for detecting hepatitis C virus target polynucleotide, its sequence is: 5'-CCAUCUGCGGAACCGGUGAGAUGG-3' (sequence 5), the 5'end is fluorescently labeled with FAM, and the 3'end is f...

Embodiment 2

[0131] Example 2 Preparation of hepatitis C virus ( HCV ) Real-time fluorescent nucleic acid constant temperature amplification detection kit

[0132] Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for hepatitis C virus of the present invention is obtained. The kit includes capture probe (TCO, TargetCapture Oligo), T7 primer, nT7 primer, detection probe, M-MLV reverse transcriptase and T7 RNA polymerase, among which:

[0133] The capture probe nucleotide sequence is SEQ ID NO: 2, the T7 primer sequence SEQ ID NO: 3, the nT7 primer sequence is SEQ ID NO: 4, and the detection probe nucleotide sequence is SEQ ID NO: 5.

[0134] The capture probe is present in the viral nucleic acid extraction solution, the T7 primer, nT7 primer and HCV detection probe are present in the HCV amplification detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase are present in SAT enz...

Embodiment 3

[0152] Example 3 Real-time fluorescent nucleic acid constant temperature amplification detection of hepatitis C virus nucleic acid national reference product

[0153] Use the kit in Example 2 to test the negative coincidence rate, positive coincidence rate, and sensitivity of the national reference product of hepatitis C virus nucleic acid of the China Food and Drug Control Institute in a 10-fold serial dilution to 50 IU / ml (10 repeated tests) ) Perform real-time fluorescent nucleic acid constant temperature amplification detection, where the positive reference product is P1-P10, and the negative reference product is N1-N10; a negative control and a positive control (10 6 Copy / ml dilution of HCVICRNA) one each, the specific steps are as follows:

[0154] 1. Nucleic acid extraction

[0155] Add 400 μl of viral nucleic acid extract (HEPES500mM, LLS8%, capture probe 15μM, magnetic beads 150mg / L) and 250μl of reference sample into the sample processing tube, mix well, incubate at 60°C f...

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Abstract

The invention discloses a real-time fluorescence nucleic acid isothermal amplification detection kit for HCV (hepatitis C virus). The detection kit specifically comprises a capture probe, HCV primers, an HCV detecting probe, M-MLV reverse transcriptase, T7RNA polymerase and the like, wherein the HCV primers include a T7 primer and an nT7 primer. By the detection kit which is high in detection efficiency, high in accuracy and promising in application prospect, nucleic acid amplification detection can be performed on serum or plasma samples containing hepatitis C viruses in a high-specificity, high-sensitivity, low-pollution and fast manner, and 6 genotypes of HCV can be detected.

Description

technical field [0001] The invention relates to the technical field of biomedical detection of viruses, in particular to primers and probes used in the real-time fluorescent nucleic acid constant temperature amplification detection of hepatitis C virus HCV that combines specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology and related kits. Background technique [0002] Hepatitis C virus, referred to as hepatitis C, hepatitis C, is a kind of viral hepatitis caused by hepatitis C virus (Hepatitis C virus, HCV) infection, mainly through blood transfusion, acupuncture, drug abuse, etc., according to the statistics of the World Health Organization , the global HCV infection rate is about 3%, it is estimated that about 180 million people are infected with HCV, and there are about 35,000 new cases of hepatitis C every year. Hepatitis C is a global epidemic, which can lead to chronic inflammation, necrosis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 尹华立于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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