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Application of crRNA targeted PCR-CRISPR system to detection of HBVDNA

A system and purpose technology, applied in the direction of recombinant DNA technology, DNA/RNA fragment, microbial determination/inspection, etc., can solve the problems of high cost, prone to contamination, not suitable for clinical detection, etc., to achieve simple method, high sensitivity and specific effect

Active Publication Date: 2019-08-30
中国人民解放军疾病预防控制中心 +1
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  • Summary
  • 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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  • Application of crRNA targeted PCR-CRISPR system to detection of HBVDNA
  • Application of crRNA targeted PCR-CRISPR system to detection of HBVDNA
  • Application of crRNA targeted PCR-CRISPR system to detection of HBVDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: be used for the design and preparation of crRNA of the present invention and PCR primer

[0041] (1) Design and preparation of crRNA

[0042] The detection target of HBV DNA is selected near the P area of ​​hepatitis B virus. First, the conservative analysis of this part of the sequence was carried out, and the hepatitis B virus database ( https: / / hbvdb.ibcp.fr ) to download the sequence information of the P region of 7720 strains of hepatitis B virus, apply clustal X software to carry out bioinformatics analysis on the sequence, and use the Perl script to analyze the base ratio of each site. Then select and design corresponding PCR amplification primers and crRNA according to the analysis results.

[0043] The present invention selects the conserved sequence of the reverse transcriptase region (RT region) to design crRNA. The 5' end of the crRNA has a 39nt repeat sequence, which can bind to LwCas13a protein, 5'-GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-...

Embodiment 2

[0068] Example 2. Establishment of the method for detecting HBV DNA by CRISPR-Cas13a

[0069] In this study, PCR was used to amplify the target nucleic acid, and the target sequence detected by HBV DNA was transcribed to obtain the corresponding ssRNA, and the above crRNAs: HBV-crRNA1, HBV-crRNA2, HBV-crRNA3 were used for detection, and the results of different crRNAs were compared. Signal intensity, select the crRNA with the strongest fluorescent signal as the subsequent crRNA detection. Specific steps and principles such as figure 1 Shown: the first step uses specific primers to amplify the target sequence (through denaturation, annealing, and extension processes), and there is a T7 transcription sequence at the 5' end of the primer, so that the double-stranded DNA obtained by PCR amplification ( dsDNA) can be recognized and transcribed by T7 RNA polymerase. In the second step, part of the amplified product was taken out and added to T7 RNA polymerase, LwCas13a protein, cr...

Embodiment 3

[0079] Example 3. Sensitivity of CRISPR-Cas13a to detect HBV DNA

[0080] The sensitivity experiment mainly detects the lowest copy number of hepatitis B virus that can be detected by this method, and compares it with the existing fluorescent quantitative PCR detection method. The plasmid pHBV1.2 was serially diluted to 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 Copies / ul, after PCR amplification, take 5ul of the amplified product and add it to the LwCas13a detection system, and set the PCR product with water as the template as a negative control. And take 2ul PCR products for agarose gel electrophoresis detection. At the same time, the hepatitis B virus DNA detection kit (Hunan Shengxiang Biotechnology Co., Ltd.) was used to detect different concentrations of plasmid templates, and the sensitivity of the two detection methods was compared. The agarose gel results of PCR amplification products are shown in image 3 , image 3 There is no band in the negative control (N...

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Abstract

The invention discloses a method for detecting HBVDNA through combination of PCR and CRISPR. The method comprises the steps of (1) amplifying nucleic acid of samples to be detected through a pair of upstream and downstream specific primers, wherein a sequence which can be recognized and transcribed by T7RNA polymerase is arranged at a 5' end of the downstream primer; and (2) detecting whether a targeting sequence exists or not in amplification products of the nucleic acid of the samples to be detected in a detection system containing crRNA, T7RNA polymerase, Cas13a protein and report RNA for recognizing an HBVDNA target sequence, wherein the section of the HBVDNA target sequence is 803<rd>-829nt. The invention further discloses crRNA capable of targeting HBVDNA specific sites and a reagent kit containing the crRNA. The method provided by the invention is simple, convenient and quick during detection of the content of the HBVDNA, and has extreme high sensitivity and specificity, single-copy HBV DNA is differentiated within 15 minutes after PCR amplification, and higher positive detection rate can be shown for serum samples low in virus load.

Description

technical field [0001] The invention relates to a crRNA sequence and a technology for detecting hepatitis B virus gene 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. It can be seen that early detection and early treatment of HBV are very important for HBV patients. [0003] HBV DNA is the core substance of hepatitis B virus and the basis of virus replication. Under the action of the genetic gene carried...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/706C12Q1/686C12Q2521/327C12Q2521/119C12Q2563/107
Inventor 宋宏彬李浩王珊郝荣章邱少富寇志华周育森董雪
Owner 中国人民解放军疾病预防控制中心
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