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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based PCR (Polymerase Chain Reaction) typing method and application thereof

A reaction and reaction reagent technology, applied in the field of CRISPR typing PCR, can solve the problems of false positives, non-specific amplification, and weak primer specificity in PCR detection

Active Publication Date: 2018-09-04
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In application, the main problems faced by PCR technology are the design of specific PCR primers and the non-specific amplification caused by primer specificity; these problems make PCR detection prone to false positives

Method used

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  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based PCR (Polymerase Chain Reaction) typing method and application thereof
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based PCR (Polymerase Chain Reaction) typing method and application thereof
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based PCR (Polymerase Chain Reaction) typing method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Schematic diagram of the principle and flow chart of ctPCR3.0 detection and typing of DNA molecules figure 1 shown. Schematic diagram of ctPCR3.0 detection of DNA molecules ( figure 1 ). The method detects target DNA homogeneously in one step: the detected DNA sample is first cleaved by a pair of Cas9 / sgRNA complexes, followed by qPCR with a pair of universal primers. All detection components (DNA to be detected, Cas9 protein, sgRNA and qPCR reagents) are pre-mixed in one PCR tube. The whole detection process is to add a period of constant temperature incubation time (37° C., 30 minutes) before the common PCR procedure. During the constant temperature incubation step, Cas9 nucleases complexed with a pair of sgRNAs (sgRNAa and b) respectively and cut DNA samples simultaneously. Specific cleavage of target DNA by Cas9 / sgRNA will result in increased Ct values ​​in qPCR amplification.

Embodiment 2

[0045] Cutting HPV plasmids with Cas9 / sgRNA and identifying HPV16 and 18 with ctPCR3.0

[0046] experimental method:

[0047] Preparation of sgRNA:

[0048] Preparation of sgRNA in vitro transcription template: PCR1: According to the backbone part of sgRNA, first design a pair of primers (F1 and R as shown in Table 1) for PCR. PCR reaction system (30 μL): 2 μL F1 (Table 1), 2 μL R (Table 1), 15 μL 2×primestar (TAKARA), with H 2 O Make up the volume to 30 μL. PCA reaction program: 95°C for 2 minutes; 7 cycles: 95°C for 15 seconds, 72°C for 1 minute. Then use 1.5% agarose gel 100V electrophoresis for 40 minutes, recover and purify with gel recovery kit (Axygen), dissolve in 25 μ L of eluent, and use Nanodrop2000 spectrophotometer to detect its DNA concentration and purity, and this fragment is named fragment 1. Store at -20°C for later use. PCR2: PCR amplification was carried out using fragment 1 as a template and F2 and Sg-R as primers. PCR reaction system (50 μL): 2 μL F...

Embodiment 3

[0067] Detection of L1 gene in HPV subtype plasmid by ctPCR3.0

[0068] experimental method:

[0069] HPV plasmid DNA (2ng) was cleaved by sgRNAs / Cas9 nuclease and directly entered into qPCR reaction. ctPCR3.0 reaction (30 μL): 15 μL 2×SYBR Green Master Mix (Yeasen), 1 μM Cas9 nuclease (NEB), 300 nM sgRNAa (Table 2), 300 nM sgRNAb (Table 2), 500 nM L1-MY09 (Table 3), 500 nM L1-MY11 (Table 3) and 2ng HPV plasmid DNA. Run protocol: 37°C for 30 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 58.5°C for 30 seconds and 72°C for 45 seconds. The reaction was carried out on the real-time PCR device StepOne plus (ABI).

[0070] Experimental results:

[0071] In order to further verify the specificity of ctPCR3.0. There are 10 subtypes of HPV (high risk: 16, 18, 33, 35, 45, 51, 52, 56, 58, and 59) L1 plasmid DNA, and 10 pairs of sgRNAs are combined with Cas9 to cut each subtype HPV plasmid DNA (37°C, 30 minutes). The Cas9 protein was then inactivated at 95°C while...

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Abstract

The invention discloses a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based PCR (Polymerase Chain Reaction) typing method and the application thereof. The method is a method forperforming DNA detection and typing. The method comprises the following steps: adding a Cas9 protein and sgRNA for targeting target DNA into a conventional PCR reaction system, adding a short-term thermostatic incubation program before the PCR reaction program, and starting a conventional PCR amplification program. The CRISPR based PCR typing method disclosed by the invention adopts a homogeneousdetection technology, and detection can be completed by a PCR amplification step only. By utilizing a specific recognition cutting characteristic of the CRISPR technology on the DNA, the target DNA can be simply, homogeneously, rapidly and sensitively subjected to specific detection and typing, and the method is a novel DNA detection method with high specificity and sensitivity. According to themethod in the invention, human HPV DNA in clinical samples can be successfully detected.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to a CRISPR-based DNA detection and typing method, specifically a CRISPR typing PCR method (ctPCR method for short) and its application. Background technique [0002] DNA testing and genotyping have always been important for basic research and various testing and diagnostic applications. Therefore, DNA detection and genotyping technology has been receiving widespread attention, thus promoting the development of this type of technology. In short, there are three main types of DNA testing and genotyping technologies that are widely used. The first are various techniques based on polymerase chain reaction (PCR). PCR is the most commonly used DNA detection and genotyping technology. PCR-based DNA detection and genotyping mainly rely on the design of specific primers and multiplex PCR amplification. PCR detection can be achieved by traditional PCR (tPCR), quantitative PCR (qPCR) and the rec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2521/301
Inventor 王进科张贝贝
Owner SOUTHEAST UNIV
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