Detection kit of hepatitis B virus resistance to drug and hepatitis B virus genotype and detection method using the same

A hepatitis B virus and kit technology, applied in the biological field, can solve the problems of easy misjudgment in naked eye interpretation, poor scalability and low throughput of linear probe reverse hybridization

Active Publication Date: 2016-03-30
BOAO BIOLOGICAL CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(1) Sequencing is the gold standard for HBV gene detection, with high accuracy, but low sensitivity, poor ability to detect mixed genotypes (need to screen a large number of clones), requires a sequencer and expensive reagents, and is not suitable for clinical detection; (2) RFLP method is not suitable for clinical detection. Complex equipment and reagents are required, but the standardization degree of the current method is poor; (3) the quantitative PCR method has high sensitivity, but the throughput is low, and it is difficult to detect the variation of multiple gene loci; Interpretation is easy to misjudge, the cost of detection is ...

Method used

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  • Detection kit of hepatitis B virus resistance to drug and hepatitis B virus genotype and detection method using the same
  • Detection kit of hepatitis B virus resistance to drug and hepatitis B virus genotype and detection method using the same
  • Detection kit of hepatitis B virus resistance to drug and hepatitis B virus genotype and detection method using the same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Embodiment 1, detection kit and detection method

[0111] 1. Kit composition

[0112] The kit of the invention is composed of primers, PCR reaction mixture, gene chip, hybridization reagent, negative control substance and positive control substance.

[0113] 1. Primers

[0114] The invention adopts various types of specific primers with Tag tag sequences and paired primers with fluorescent labels to carry out multiple asymmetric PCR amplification and fluorescent labeling for each drug-resistant mutant type and genotype of HBV.

[0115] The primers for multiple asymmetric PCR amplification are specific primers with specific tag sequences, which can specifically combine with target HBV gene sequences. An oligonucleotide sequence that has nothing to do with the primer amplification template sequence is added to the 5' end of the primer. The oligonucleotide sequence can specifically bind to the probe immobilized on the chip, and the 5' end of the primer paired with the sp...

Embodiment 2

[0180] Embodiment 2, the sensitivity detection of hepatitis B virus DNA

[0181] The plasmid (concentration of about 10 3 IU / ml) is a template, and the reagent of the present invention is used to detect, and the detection results are shown in the appendix Figure 1-Figure 10 .

[0182] from Figure 1-Figure 10 As can be seen from the results, the method and kit of the present invention can detect that the concentration is about 10 3 The common 7 mutant types and 3 genotype HBV DNA templates in IU / ml can meet the clinical needs.

Embodiment 3

[0183] Embodiment 3, the specific detection of hepatitis B virus DNA

[0184] The purchased genome DNA (human genome DNA, herring sperm DNA and Escherichia coli DNA) that has nothing to do with hepatitis B virus outside the detection range of the kit is used as a template, and the concentration is about 10ng / μl, which is detected using the kit of the present invention , see the test results in the appendix Figure 11-Figure 13 .

[0185] from Figure 11-Figure 13 As can be seen from the results, the method and kit of the present invention are negative for the DNA template irrelevant to hepatitis B virus at a concentration outside the detection range of the kit of 10 ng / μl, indicating that the specificity of the kit is good.

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Abstract

The invention discloses a detection kit of hepatitis B virus resistance to a drug and a hepatitis B virus genotype and a detection method using the same. The kit is used based on a gene chip and contains three groups of primers. In detection, the three groups of primers respectively carry out multiple asymmetric PCR amplification processes on a sample to be detected, the three PCR amplification products are mixed, the mixture and a hybridization buffer solution are mixed to form a hybridization solution, the hybridization solution and a probe of the gene chip are subjected to hybridization and a mutant type and a genotype of the sample to be detected are determined through a fluorescence signal of the PCR product. The detection result shows that the kit has good specificity and high sensitivity and can simultaneously detect seven mutant types and three genotypes of samples to be detected. The seven mutant types comprise a 180M type, a 181T type, a 181V type, a 204I type, a 204V type, a 236T type and a 1896A type and the three genotypes comprise a HBV/B type, a HBV/C type and a HBV/D type.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit and detection method for drug resistance and genotype of hepatitis B virus. Background technique [0002] Hepatitis B virus is a hepatotropic DNA virus, which is one of the main pathogens causing acute and chronic hepatitis, and can lead to liver cirrhosis, liver failure and liver cancer. my country is a country with a high incidence of hepatitis B. In 2009, the bulletin of the Ministry of Health showed that the incidence of hepatitis B in 2008 was 1.0654 / 100,000, and the number of cases was 1.41 million. HBV infection is a threat to national health, and the early diagnosis and correct treatment of HBV should receive great attention. [0003] Interferon, lamivudine, telbivudine, adefovir and the soon-to-be-launched tenofovir are all first-line antiviral drugs in China. Strict correction mechanism leads to high nucleotide mismatch rate during HBV replication. Gene mu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 孙兰华刘莹莹张冠斌项光新邢婉丽
Owner BOAO BIOLOGICAL CO LTD
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