A kind of method and application of CRISPR typing PCR
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, and achieve the avoidance of nucleic acid hybridization and specific PCR primer design, The effect of sensitive and specific detection and typing
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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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