Novel coronavirus nucleic acid detection kit

A detection kit and coronavirus technology, applied in the direction of measuring devices, microbe determination/inspection, and resistance to vector-borne diseases, etc., can solve the problems of undiagnosed, fast transmission, lag, etc., and achieve fast detection speed and simple method , low cost effect

Active Publication Date: 2020-07-24
武汉博杰生物医学科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus isolation method takes a long time and cannot be widely used in clinical rapid and accurate detection; immunological methods (ELISA, antigen antibody detection, etc.) can be quickly detected, but the sensitivity is insufficient, and it can only be detected after illness, which is relatively lagging. It is not conducive to screening and can not be diagnosed; modern molecular biology diagnostic methods have good specificity and high sensitivity, and most of the detection kits on the market use RT-PCR method
As a newly discovered virus, the new coronavirus coincides with the Spring Festival travel season, the peak flow of people, and the rapid spread. The time-consuming preparation cycle of conventional virus isolation methods and immunological methods for specific antibodies makes it unable to meet the clinical needs of many suspected patients. testing needs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides a novel coronavirus nucleic acid detection kit. The novel coronavirus nucleic acid detection kit comprises two crDNAs or two crRNAs. The crDNAs comprise a crDNA of a targeting Egene and a crDNA of a targeting N gene, wherein the sequence of the crDNA of the targeting E gene is as shown in any one of SEQ ID NO.3-5, the sequence of the crDNA of the targeted N gene is shown asany one of SEQ ID NO.6-8. The crRNAs comprise a crRNA of the targeting E gene and a crRNA of the targeting N gene, wherein the sequence of the crRNA of the targeting E gene is as shown in any one ofSEQ ID NO.9-11, and the sequence of the crRNA of the targeting N gene is as shown in any one of SEQ ID NO.12-14. The crRNAs related to the novel coronavirus E gene and the novel coronavirus N gene aredesigned, and screening detection is carried out in combination with a CRISPR-cpf1 system. The method has the advantages of being simple, high in speed, high in sensitivity, high in specificity and extremely high in practicability.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a novel coronavirus nucleic acid detection kit. Background technique [0002] Current virus detection methods mainly include virus isolation methods, immunological methods and molecular diagnostic methods. The virus isolation method takes a long time and cannot be widely used in clinical rapid and accurate detection; immunological methods (ELISA, antigen antibody detection, etc.) can be quickly detected, but the sensitivity is insufficient, and it can only be detected after illness, which is relatively lagging. It is not conducive to screening and cannot be diagnosed; modern molecular biology diagnostic methods have good specificity and high sensitivity, and most of the detection kits using RT-PCR method are currently on the market. As a newly discovered virus, the new coronavirus coincides with the Spring Festival travel season, the peak flow of people, and the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/70C12Q1/6844G01N33/543G01N33/558G01N33/569
CPCC12N15/113C12Q1/6844C12Q1/701G01N33/54313G01N33/558G01N33/56983G01N2333/165C12Q2521/107C12Q2563/107Y02A50/30
Inventor 姚杰程诚王恩慧陈威巍许文杨俊连杨欣欣李胜
Owner 武汉博杰生物医学科技有限公司
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