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PCR-CRISPR detection method for targeting HBV drug-resistant mutant gene

A PCR-CRISPR and drug-resistant gene technology, applied in the field of molecular biology, can solve the problems of high cost, easy pollution, unsuitable for clinical detection, etc., and achieve the effect of strong fluorescence detection signal and simple method

Active Publication Date: 2019-09-03
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, considering the high cost of RPA isothermal amplification technology and the fact that the amplification process is prone to contamination, it is not suitable for routine clinical testing at present.

Method used

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  • PCR-CRISPR detection method for targeting HBV drug-resistant mutant gene
  • PCR-CRISPR detection method for targeting HBV drug-resistant mutant gene
  • PCR-CRISPR detection method for targeting HBV drug-resistant mutant gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: be used for the design and preparation of crRNA of the present invention and PCR primer

[0057](1) Design of YVDD and YIDD drug-resistant mutant crRNA

[0058] A total of 32bp (725-756bp) nucleotides in the upstream and downstream of the YMDD region were analyzed for sequence conservation (see figure 2 ), for the less conserved sites, select the base with the highest probability of each site to design crRNA. figure 2 The middle box marks the rt204 site, and the analysis results show that, except for the three sites nt732, nt735 and nt753, the sequences of other sites are relatively conserved. According to the conservation characteristics of this partial sequence, we designed corresponding crDNA templates and PCR primers (see Table 2) for preparing crRNA for mutation detection. The DNA sequence was synthesized by Beijing Tianyi Huiyuan Company.

[0059] Table 2. crDNA templates and PCR primers used to prepare crRNA for mutation detection

[0060] ...

Embodiment 2

[0084] Embodiment 2.Specific detection of YVDD and YIDD drug-resistant mutation crRNA

[0085] The target sequences of the wild strain of hepatitis B virus and the two drug-resistant mutant strains were transcribed into corresponding ssRNA, and the specificity of the three sequences were detected by using the above-mentioned two drug-resistant mutant crRNAs.

[0086] (1) Detection of mutant crRNA to wild-type and mutant sequences

[0087] Using the wild-type and two drug-resistant mutant plasmids constructed above and the designed primers HBV-F and HBV-R, amplification, transcription, and purification of ssRNA were carried out. The reaction system and conditions were the same as before. Two kinds of crRNA were used to detect ssRNA of three sequences respectively, and the detection system was prepared as follows:

[0088] Table 6. ssRNA Target Cutting System

[0089]

[0090]

[0091] Then, at 37°C, the fluorescent signal of the FAM channel detection system of the fluor...

Embodiment 3

[0103] The sensitivity detection of embodiment 3.YMDD drug resistance mutation

[0104] (1) Agarose gel electrophoresis detection

[0105] YVDD, YIDD mutant plasmids and YMDD wild-type plasmid standard products were serially diluted and used as templates for PCR amplification, followed by 1.5% agarose gel electrophoresis detection, and the electrophoresis results were as follows: Figure 8 shown. Figure 8 middle. A. The electrophoresis results of the HBV WT wild-type plasmid gradient dilution PCR, B. the electrophoresis results of the HBV YIDD mutant plasmid gradient dilution PCR, and C. the electrophoresis results of the HBV YVDD mutant plasmid gradient dilution PCR. The negative control (NC) corresponding to the three groups of templates had no bands, indicating that there was no pollution during the amplification process; the results showed that a single band with the same size as the target band (304bp) appeared between 250-500bp; at the same time, the three Group temp...

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Abstract

The invention discloses a PCR-CRISPR detection method for targeting a HBV drug-resistant mutant gene. The method comprises the following steps: (1) performing PCR amplification on nucleic acid of a sample to-be-tested with a pair of specific primers, wherein a 5' end of an upstream primer is provided with a sequence recognizable and transcribed by T7 RNA polymerase; and (2) detecting whether the drug-resistant mutant gene is in an amplification product of the sample nucleic acid to-be-detected in a detection system including crRNA, T7 RNA polymerase, Cas13a protein, and an RNase reporter molecule that recognizes a HBV YMDD-resistant mutation site, and a target sequence of the drug-resistant mutant gene is a YIDD or YVDD gene mutation of a YMDD region of a HBV genome. The present inventionalso discloses crRNA capable of targeting a YIDD or YVDD resistance gene mutation of HBV and a kit containing the same. The method provided by the invention can detect HBV drug-resistant mutant gene simply and quickly, and has extremely high sensitivity and specificity.

Description

technical field [0001] The invention relates to a crRNA molecule and a technology for detecting hepatitis B virus drug-resistant mutation genes through a CRISPR-Cas13a system, belonging to the technical field of molecular biology. Background technique [0002] Hepatitis B is a serious infectious disease caused by Hepatitis B Virus (HBV). About 240 million people in the world are carriers of hepatitis B virus surface antigen, and about one million people die of liver-related diseases caused by HBV every year. my country is a big country with hepatitis B, and there are about 90 million HBV infected people, including about 28 million chronic hepatitis B patients, which shows that hepatitis B virus infection has become a major problem that endangers public health. [0003] The continuous replication of hepatitis B virus is the root cause of hepatitis B disease, and the fundamental purpose of hepatitis B treatment is to inhibit virus replication. Commonly used hepatitis B antiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11C12R1/93
CPCC12Q1/706C12Q1/6858C12Q2600/156C12Q2600/106C12Q2531/113C12Q2521/327C12Q2521/119C12Q2563/107Y02A50/30
Inventor 李浩宋宏彬王珊郝荣章邱少富寇志华周育森董雪
Owner ACADEMY OF MILITARY MEDICAL SCI
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