A CRISPR nucleic acid detection kit for detecting novel coronavirus (2019-ncov)
A coronavirus and kit technology, applied in the field of CRISPR nucleic acid detection kits, can solve the problems of insufficient sensitivity, easy contamination, and high instrument requirements
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Embodiment 1
[0064] Example 1. Novel coronavirus nucleic acid detection kit and detection method based on CRISPR-Cas13a system 1. Novel coronavirus nucleic acid detection kit based on CRISPR-Cas13a system
[0065] (1) Preparation of mRNA standards
[0066] The N gene of the new coronavirus is sequence 3, wherein the 992-1019th position of sequence 3 is the target sequence of COVID-19crRNA.
[0067] The mRNA is transcribed from the PCR amplification product of the above-mentioned plasmid to simulate viral nucleic acid. The specific method steps are as follows:
[0068] (1) PCR amplification
[0069] Using the artificially synthesized DNA molecule shown in Sequence 3 as a template, PCR amplification was performed using the system shown in Table 1 below to obtain a PCR product.
[0070] The PCR reaction system was prepared as shown in Table 1.
[0071] Table 1. PCR amplification system
[0072] name volume DNA molecule shown in sequence 3 2μL nCoVnp-F1 2μL n...
Embodiment 2
[0139] Example 2, Condition optimization and exploration of detection method based on CRISPR-Cas13a system
[0140] 1. Screening of optimal RT-RAA amplification primers
[0141] Using the mRNA standard substance prepared in one (one) of Example 1 as a template, carry out RT-RAA amplification with the method of (two) 1 of Example 1, and primers are respectively each primer combination shown in Table 3 ( Combination form such as figure 2 Shown), the RT-RAA amplification product was obtained.
[0142] Set water as the template amplification product as a negative control.
[0143] Take 20 μL of the amplified product, add 20 μL of chloroform: Tris balanced phenol = 1:1 and mix with the product after the reaction, shake and centrifuge for 10 minutes, discard the supernatant mixture, shake and mix, and centrifuge at 10,000 rpm for 10 minutes , Aspirate 10 μL of the supernatant, add 3 μL 6*Loading Buffer, mix well and perform agarose gel electrophoresis.
[0144] detect as figu...
Embodiment 3
[0155] Example 3, Sensitivity detection of the detection method of novel coronavirus nucleic acid based on CRISPR-Cas13a system
[0156] The mRNA standard prepared in (1) of Example 1 was subjected to 10-fold serial dilution with RNase-free water to obtain solutions containing different concentrations of novel coronavirus gene mRNA, which were detected according to the method in Example 1, II. The RT-RAA product in Table 7 was replaced with solutions containing different concentrations of novel coronavirus gene mRNA, so that the concentration of mRNA in the reaction system was 10 5 -10 -1 copies / test, and set water as the template amplification product as a negative control.
[0157] Test results such as Figure 4 shown, 10 5 -10 1 The "T" line disappears and the "C" line appears in the copies / test group; 10 0 copies / test group, 10 -1 Both the "T" line and the "C" line of the copies / test group and the negative control group are displayed. Judgment 10 5 -10 1 Copies / ...
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