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HBV (hepatitis B virus) real-time fluorescent nucleic acid isothermal amplification detection kit

A hepatitis B virus, constant temperature amplification detection technology, applied in the field of probes, related kits, and primers, can solve the problems of inability to detect nine HBVA-I genotypes, poor specificity and sensitivity, etc. Ease of automation, broad coverage, and reduced amplification and detection time

Active Publication Date: 2016-04-27
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, this kit also solves the technical problem that the hepatitis B detection kits in the prior art have poor specificity and sensitivity and cannot detect all nine genotypes of HBVA-I

Method used

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  • HBV (hepatitis B virus) real-time fluorescent nucleic acid isothermal amplification detection kit
  • HBV (hepatitis B virus) real-time fluorescent nucleic acid isothermal amplification detection kit
  • HBV (hepatitis B virus) real-time fluorescent nucleic acid isothermal amplification detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0124] Embodiment 1, be used for the design of the special primer and probe of real-time fluorescent nucleic acid constant temperature amplification detection hepatitis B virus HBV

[0125] The present invention selects the hepatitis B virus surface antigen S gene (its target nucleotide sequence is shown in sequence 1 in the sequence table) for primer probe design, and obtains the following specific sequence:

[0126] (1) nT7 primer and T7 primer for amplifying the hepatitis B virus target polynucleotide, the nucleotide sequence of the T7 primer is: 5'-GATGTGTCTGCGGCGTTTTA-3' (sequence 1); the nucleotide sequence of the nT7 primer is: 5'-AATTTAATACGACTCACTATAGGGAGAACAAACGGGCAACATACCTT-3' (SEQ ID NO: 2);

[0127] (2) Detection probe for detecting hepatitis B virus target polynucleotide, its sequence is: 5'-CCAUCCUGCUAUGCCUCGAUGG-3' (sequence 3), the 5' end is labeled with FAM fluorescence, and the 3' end is labeled with DABCYL fluorescence .

[0128] During the design proce...

Embodiment 2

[0133] Embodiment 2 prepares hepatitis B virus ( HBV ) real-time fluorescent nucleic acid constant temperature amplification detection kit

[0134] Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for hepatitis B virus of the present invention was obtained. The kit contains capture probes (TCO, TargetCaptureOligo), T7 primers, nT7 primers, detection probes, internal standard detection probes, internal standards, M-MLV reverse transcriptase and T7 RNA polymerase and other components, of which:

[0135] The capture probe nucleotide sequence is SEQIDNO:4, the T7 primer sequence is SEQIDNO:1, the nT7 primer sequence is SEQIDNO:2, the detection probe nucleotide sequence is SEQIDNO:3, and the internal standard detection probe nucleotide sequence is SEQIDNO: 7.

[0136] The capture probe exists in the viral nucleic acid extraction solution, the T7 primer, nT7 primer, HBV detection pr...

Embodiment 3

[0156] Example 3 Real-time fluorescent nucleic acid constant temperature amplification detection of clinical serum and plasma samples

[0157] Use the kit of the present invention (see Example 2 for the composition of the capture probe, primer pair, detection probe, and internal standard detection probe) to detect hepatitis B virus in clinical serum and plasma samples, and compare it with conventional PCR to detect the same Serum samples. The specific method of SAT detection includes the following steps:

[0158] 1. Sample collection, transportation and storage

[0159] The clinician will collect the specimens according to the actual situation. The serum sample collection method is as follows: use a sterile syringe to extract 2 mL of venous blood from subjects 1-16, collect it in a sterile collection tube, and centrifuge at 1600 rpm for 5 hours at room temperature for no more than 4 hours. The serum was separated within minutes and transferred to another sterile centrifuge...

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Abstract

The invention discloses an HBV (hepatitis B virus) real-time fluorescent nucleic acid isothermal amplification detection kit, comprising a capture probe, an HBV primer, a primer T7 and a primer nT7, an HBV detection probe, an M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase, polymerase T7RNA and the like reagents. A method of the invention enables high-specificity high-sensitivity low-pollution quick nucleic acid amplification detection for serum or plasma samples containing hepatitis B virus, has the advantages of high detection efficiency and high accuracy, can detect A-I 9 genotypes of HBV and has a promising application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedical detection of viruses, in particular to primers and probes used in nucleic acid constant temperature amplification detection of hepatitis B virus (HBV), which combines specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology and related kits. Background technique [0002] Hepatitis B virus (hepatitisBvirus, HBV) infection is a serious public health problem. About 1 million people worldwide die from liver failure, liver cirrhosis and primary hepatocellular carcinoma (liver cancer) caused by HBV infection every year. More than 75% of liver cancer patients are caused by HBV. [0003] my country is an endemic area of ​​HBV infection. According to the national hepatitis B epidemiological survey in 2006, there are about 93 million chronic HBV infected people in my country, of which about 20 million are chronic hepatitis B patie...

Claims

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Application Information

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