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Hepatitis B virus genotyping detection method based on CRISPR/Cas13a system

A hepatitis B virus and genotyping technology, which is applied in the field of medical biology, can solve the problems of low detection sensitivity, difficulty, and poor stability, and achieve the effects of improving detection specificity, saving time and cost, and eliminating the need for operating procedures

Active Publication Date: 2021-02-19
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the stability of HBV genotypes detected by the linear probe method is poor, and different genotypes are distinguished only by the principle of complementary base pairing. The detection results are easily interfered by objective factors such as reaction buffer and pH value, and the detection sensitivity is low. Low, it is difficult to detect a small number of samples, and it is not suitable for the needs of clinical rapid detection
Although the HBV genotyping method of PCR technology has the advantage of high sensitivity, it requires a special thermal cycler and related technical personnel to operate, and the design of related probes is relatively complicated, so it is not suitable for on-site rapid detection in technically poor areas
The restriction fragment length polymorphism (RFLP) method has low cost and simple principle, but its sensitivity is poor and the operation process is cumbersome, which greatly limits its clinical application
The cost of typing and testing by direct sequencing of HBV genomic DNA is high, and it is not suitable for the medical needs of patients in developing countries, and the technical requirements are too high, so it is not suitable for most testing scenarios

Method used

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  • Hepatitis B virus genotyping detection method based on CRISPR/Cas13a system
  • Hepatitis B virus genotyping detection method based on CRISPR/Cas13a system
  • Hepatitis B virus genotyping detection method based on CRISPR/Cas13a system

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Design of RPA Primers for Genomic DNA of HBV Virus

[0052] 1. Use the NCBI Primer Blast online tool to design universal recombinant polymerase amplification (RPA) primers for HBV genomic DNA, which are effective for both HBV type B and C viruses, that is, only this pair of RPA primers is needed Amplification of HBV type B and C viruses.

[0053] 2. After screening, the final RPA primer pair used in the present invention is as follows: the sequence of the forward primer is shown in SEQ ID NO:1 and the sequence of the reverse primer is shown in SEQ ID NO:2.

Embodiment 2

[0055] Design crRNA sequences and synthesize them by in vitro transcription

[0056] 1. First synthesize a DNA sequence complementary to crRNA as a template for in vitro transcription. It should be noted that a complementary sequence of the T7 promoter should be extended at the 3' end of the DNA sequence, as shown in SEQ ID NO: 13, which is the subsequent T7 Prepare for in vitro transcription.

[0057] 2. Then anneal the DNA template and a short T7 promoter sequence, as shown in SEQ ID NO: 14, in 1x annealing buffer (100mM Tris-HCl (pH 7.5), 10mM EDTA, 1M NaCl), Incubate first at 95 °C for 10 min, then allow to cool slowly to room temperature.

[0058] 3. After the annealing reaction, use the HiScribe T7 Rapid High-yield RNA Synthesis Kit to incubate overnight at 37°C for in vitro transcription of crRNA. The transcription reaction contained NTP buffer mixture (the final concentration of each NTP was 10 mM), T7 RNA polymerase mixture, template DNA (2 μg), mouse RNase inhibito...

Embodiment 3

[0061] Synthesis and purification of HBV RNA

[0062] 1. First, use the designed RPA primer pair to amplify the plasmid containing HBV DNA through the RPA kit. The amplified product was then incubated overnight at 37°C for in vitro transcription using the HiScribe T7 Rapid High Yield RNA Synthesis Kit.

[0063] 2. After the in vitro transcription reaction is completed, add an appropriate amount of RNase-free DNase I (4 units in total) to the system and incubate at 37°C for 15 minutes to digest the DNA template.

[0064] 3. The obtained HBV in vitro transcription product was purified by MEGAclear transcription purification kit, and then the concentration was measured by Nanodrop, and verified by denaturing urea polyacrylamide gel electrophoresis (urea / PAGE).

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Abstract

The invention belongs to the field of medical biotechnology research, and discloses a hepatitis B virus genotyping detection method based on a CRISPR / Cas13a system. The hepatitis B virus genotyping detection method comprises the following steps of 1, designing a crRNA sequence, and carrying out in vitro transcription synthesis; 2, designing a universal recombinant polymerase amplification (RPA) primer of the HBV genome DNA by using an NCBI Primer Blast online tool; 3, synthesizing and purifying hepatitis B virus RNA; 4, using Cas13a enzyme and HBV crRNA capable of recognizing HBV B-type and C-type viruses at the same time for sensitivity testing of the detection method; and 5, carrying out enzyme digestion fluorescence test on the plasmid containing the HBV B-type or-C type genome DNA by using Cas13a enzyme and crRNA. Compared with the conventional detection means, the scheme disclosed by the hepatitis B virus genotyping detection method based on the CRISPR / Cas13a system has the characteristics of simplicity in operation, high sensitivity and low price, and meanwhile, an additional hairpin structure is contained in the specific recognition area, so that the high detection specificity is improved, and the HBV genotype can be distinguished within 1-2h.

Description

technical field [0001] The invention belongs to the field of medical biotechnology research, and relates to a hepatitis B virus genotyping detection method based on a CRISPR / Cas13a system. Background technique [0002] Hepatitis B virus (HBV) remains a public health problem worldwide, with more than 350 million infected individuals at risk of developing end-stage liver disease and hepatocellular carcinoma (HCC). China is a high prevalence area of ​​hepatitis B, as more than 8% of the population reported chronic HBV infection. HBV can be divided into 10 genotypes, labeled A to J, which are clearly distributed in nucleotide sequence. Genotypes B and C are mainly distributed in Oceania and Asia, including China, while genotypes A and D are distributed in Africa and Europe. Research evidence suggests that understanding genotype has important implications for disease progression and identifying effective antiviral treatments. For example, type B and C infections have a lower c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/706C12Q1/6844C12Q2600/156C12Q2600/112C12Q2521/507C12Q2522/101Y02A50/30
Inventor 丁显廷郅晓柯雨晴
Owner SHANGHAI JIAO TONG UNIV
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