A novel coronavirus nucleic acid detection kit
A detection kit, coronavirus technology, applied in measurement devices, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of lag, undiagnosed, fast transmission, etc., achieving low cost and fast detection speed , the effect of the simple method
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Embodiment 1
[0041] Example 1 Design and acquisition of crRNA targeting virus-specific sites
[0042] 1. Discovery of novel coronavirus detection sites based on CRISPR-cpf1 system
[0043] According to the latest "RT-PCR Detection Guidelines" issued by CDC, we obtained the specific region of the new coronavirus. Design crRNA for these different regions and construct CRISPR / cpf1 system for research. The results show that using the region sequence shown in SEQ ID NO.1-2 as the detection region based on the CRISPR / cpf1 system has a good detection effect.
[0044] Table 1
[0045]
[0046]
[0047] 2. Design of crRNA targeting specific regions
[0048] (1) Design principles of crRNA targeting specific regions
[0049] Since the CRISPR-cpf1 system is a novel targeted DNA gene editing system, in which cpf1 combines with crRNA to form a monitoring complex, the guide region of crRNA recognizes the target DNA with a complementary sequence, and cpf1 degrades the target DNA strand, the crRNA ...
Embodiment 2
[0063] Embodiment 2 Novel coronavirus fluorescent test kit and detection method
[0064] 1. The composition of the kit
[0065] (A) 2 crRNAs (crRNA targeting E gene: its sequence is as shown in any of SEQ ID NO.9-11; crRNA targeting Ngene: its sequence is as shown in any of SEQ ID NO.12-14 shown) or 2 pieces of crDNA (crDNA targeting E gene: its sequence is as shown in any of SEQ ID NO.3-5; crDNA targeting N gene: its sequence is as shown in any of SEQ ID NO.6-8 Shown. When the kit is crDNA, the operator needs to first generate RNA from the crDNA fragments under the action of T7 RNA polymerase, recover and purify to obtain crRNA);
[0066] (B) a specific fluorescent probe (see Table 3 for the sequence, any probe shown in SEQ ID NO.15-17, the 5' end of the sequence of the fluorescent probe is marked with a fluorescent group, The 3' end labels all have quenching groups; the fluorescent groups include one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the quenchin...
Embodiment 3
[0084] Colloidal gold detection kit and detection method of embodiment 3 novel coronavirus
[0085] 1. The composition of the kit
[0086] (1) The detection reagent is the same as in Example 2, except that the specific fluorescent probe in this example is any one of the probes shown in SEQ ID NO.15-17, and the 3' end of the sequence is labeled with biotin , the 5' end is marked with one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red; in this embodiment, the 5' end is marked with FAM.
[0087] (2) When the detection kit is prepared into a colloidal gold detection kit, such as Figure 5 As shown, the colloidal gold detection kit includes a base plate 1, a sample pad 2, a bonding pad 3, a nitrocellulose membrane 4 and a water-absorbing pad 7 that are bonded to the base plate 1 and overlapped successively; A quality control line 5 is provided near the side of the binding pad 3, and a detection line 6 is provided on the side of the nitrocellulose membrane 4 close to the ...
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