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CRISPR-Cas12a-based streptococcus suis rapid visual RPA detection kit and application thereof

A detection kit, Streptococcus suis technology, applied in the field of medical detection, can solve the problems of reduced reliability and achieve the effect of low equipment requirements, high sensitivity and good specificity

Pending Publication Date: 2021-11-09
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Compared with traditional PCR, LAMP detection sensitivity is high, but the addition of multiple pairs of primers in the reaction reduces the reliability and may produce false positive detection results

Method used

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  • CRISPR-Cas12a-based streptococcus suis rapid visual RPA detection kit and application thereof
  • CRISPR-Cas12a-based streptococcus suis rapid visual RPA detection kit and application thereof
  • CRISPR-Cas12a-based streptococcus suis rapid visual RPA detection kit and application thereof

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Embodiment 1

[0040] 1 Materials and methods

[0041] Streptococcus suis, Actinobacillus pleuropneumoniae used in the embodiments of the present invention, Haemophilus parasuis, Salmonella bacterial strain porcine circovirus, porcine pseudorabies virus, porcine epidemic diarrhea virus genomic DNA or cDNA are researched by Chinese Academy of Agricultural Sciences Harbin Veterinary Medicine Provided. DNA extraction kit (Beijing Tiangen P302), RNA purification and recovery kit (Beijing Tiangen, DP421), Basic (TwistDx, UK), Hisscribe T7 Quick High Yield RNA SynthesisKit (New England Biolabs), LbCas12a (New England Biolabs), MileniaHybriDetect 1Dipstick (TwistDx), centrifuge (eppendorf), constant temperature water bath (Jinghong), electrophoresis apparatus (Bio-Rad), vortex instrument (Vortex-Genie2), and nucleic acid detection imaging system (Bio-Rad), Nanodrop (IMPLEN), multi-functional microplate reader (Perkin Elmer ENSPIRE), blue light gel imager (Shanghai Sheng Work), primers and report...

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Abstract

The invention discloses a CRISPR-Cas12a-based streptococcus suis rapid visual RPA detection kit and application thereof. The kit comprises a pair of specific primer pairs for amplifying a streptococcus suis cps2j gene, gRNA and a fluorescent report probe, wherein the nucleotide sequences of the primer pairs are shown as SEQ ID NO: 6 and 7 or SEQ ID NO: 8 and 9; and the nucleotide sequence of DNA complementary to the gRNA is shown as SEQ ID NO: 2 or 3. According to the kit, specific Recombinase Polymerase Amplification (RPA) primers and a CRISPR / Cas12a system probe are designed according to the specific gene Cps2j of streptococcus suis, RPA specific target gene amplification and report gene cutting of a CRISPR / Cas12a system are realized, and a rapid visual detection method for the streptococcus suis is established. The method is rapid, visual, good in specificity, high in sensitivity and particularly suitable for detecting the streptococcus suis in resource-deficient areas.

Description

technical field [0001] The invention relates to a Streptococcus suis detection kit and its application, in particular to a CRISPR-Cas12a-based rapid visual RPA detection kit for Streptococcus suis and its application. The invention belongs to the technical field of medical detection. Background technique [0002] Streptococcus suis is the main pathogen causing swine streptococcosis, which can lead to swine lymph node abscess and septic streptococcal disease, characterized by sepsis, meningitis, arthritis, suppurative lymphadenitis and sudden death. Pigs of all ages can be infected and can occur throughout the year. With the development of my country's large-scale pig industry, suis streptococcal disease has become a common and frequently-occurring disease in pig production, especially in the past ten years, its incidence has continued to increase, which has brought great harm to my country and the world's pig industry. caused huge economic losses. S. suis is also a zoonoti...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12R1/46
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2521/327C12Q2525/151C12Q2525/161C12Q2563/107
Inventor 李刚张跃灵王春来刘思国
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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