CN-122012764-A - Detection kit and detection method for detecting Pasteurella multocida based on CRISPR/Cas12a

Abstract

The invention relates to the technical field of microorganism detection, in particular to a detection kit and a detection method for detecting Pasteurella multocida bovis based on CRISPR/Cas12 a. The invention designs a isothermal amplification primer pair based on the conserved kmt genes of the Pasteurella multocida, carries out amplification marking on kmt genes by using isothermal amplification technology, and constructs a detection kit for detecting the Pasteurella multocida by utilizing the high specific recognition capability of CRISPR/Cas12a and the visual advantage of an immune test strip. The detection kit and the detection method provided by the invention can be used for rapidly detecting the Pasteurella multocida at normal temperature and constant temperature, and have the advantages of visual and accurate result, high sensitivity, simple operation steps and no need of large-scale precise instruments. The kit provided by the invention is suitable for various field detection scenes such as farms, basic-level veterinary stations and the like, and provides a high-efficiency tool for early prevention and control of bovine pasteurellosis.

Inventors

• HAN XUESHU
• CHI LIANG
• LIU HUANQI
• WANG RUIGUO

Assignees

• 青岛农业大学

Dates

Publication Date

20260512

Application Date

20260325

Claims (10)

1. 1. A detection kit for detecting bovine pasteurella multocida based on CRISPR/Cas12a is characterized by comprising a isothermal amplification reagent, a CRISPR/Cas12a reagent and a nucleic acid detection test strip; The isothermal amplification reagent comprises a primer pair for amplifying the Pasteurella multocida kmt gene, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 4; the CRISPR/Cas12a reagent comprises crRNA, cas12a protein and a report probe, wherein the nucleotide sequence of the crRNA is shown as SEQ ID NO. 17; the nucleic acid detection test strip comprises a chromogenic substance, a T line detection line and a C line detection line.
2. 2. The test kit of claim 1, wherein the reporter probe is a 5 'FAM-labeled, 3' biotin-labeled single-stranded nucleotide sequence.
3. 3. The test kit of claim 2, wherein the single-stranded nucleotide sequence is 5'-TTATT-3'.
4. 4. The detection kit according to claim 1, wherein the chromogenic substance is colloidal gold, an anti-FAM antibody is immobilized on the T line detection line, and an anti-gold mouse antibody is immobilized on the C line detection line.
5. 5. The test kit according to claim 4, wherein the nucleic acid detection test strip further comprises a sample pad, a binding pad, an NC membrane, and a water absorbing pad; the sample pad is used for receiving a sample to be detected; The binding pad contains a colloidal gold-labeled anti-FAM antibody and a colloidal gold-labeled streptavidin; The NC film comprises a T line detection line and a C line detection line.
6. 6. Use of the detection kit of claim 1 for visually detecting pasteurella multocida.
7. 7. A method for detecting pasteurella multocida using the detection kit of claim 1, comprising the steps of: obtaining genome DNA of a sample to be tested; taking the genome DNA as a template, and carrying out isothermal amplification reaction by using the primer pair to obtain an amplification product; mixing an amplification product and a CRISPR/Cas12a reagent, and performing CRISPR/Cas12a cutting reaction at 37 ℃ for 5-30 min to obtain a reaction product; and (3) dripping the reaction product to a sample pad of the nucleic acid detection test strip, standing for 5min to 10min at room temperature, and observing a color development result, wherein the detection result is negative if the T line detection line and the C line detection line are developed, the sample to be detected does not contain the Pasteurella multocida, and the detection result is positive if the C line detection line is developed, and the sample to be detected contains the Pasteurella multocida.
8. 8. The method according to claim 7, wherein each 20. Mu.L of the cleavage reaction system comprises 2. Mu.L of 10 XCas 12a buffer, 1. Mu.L of 1. Mu.M Cas12a protein, 1. Mu.L of 600 nM-1. Mu.M crRNA, 1. Mu.L of 1. Mu.M reporter probe, 2. Mu.L of amplification product, and 13. Mu.L of enzyme-free water.
9. 9. The method according to claim 7, wherein the isothermal amplification reaction is performed at a temperature of 37-39 ℃ for 15-25 min.
10. 10. A test agent for pasteurella multocida, characterized in that the test agent is a primer pair according to claim 1.

Description

Detection kit and detection method for detecting Pasteurella multocida based on CRISPR/Cas12a Technical Field The invention relates to the technical field of microorganism detection, in particular to a detection kit and a detection method for detecting Pasteurella multocida bovis based on CRISPR/Cas12 a. Background The bovine pasteurellosis is also called bovine hemorrhagic septicemia, and the disease is caused by pasteurellosis (Pasteurella multocida), can cause symptoms such as hyperpyrexia, dyspnea, acute death and the like of the cattle, and causes serious loss to the cattle raising industry. At present, the detection of the cattle Pasteurella multocida becomes a key link of epidemic disease prevention and control, and the rapid and accurate detection is an important support for epidemic situation control, cost reduction and synergy. Traditional detection methods mainly comprise bacterial isolation and culture, serological detection and conventional molecular biological detection. However, these methods are long in time, require laboratory operations, and require specialized technicians to complete the operations, and cannot meet the requirements of rapid real-time detection of the basic level on site. The recombinase polymerase mediated isothermal amplification (RPA) is a rapid isothermal amplification (PCR) technique that allows rapid amplification of nucleic acids without the need for specialized instrumentation and higher reaction temperatures than conventional PCR techniques. However, when the detection is carried out by only the RPA technology, the sample pollution is possible, and the detection sensitivity and specificity are not high. The CRISPR/Cas12a system is a new generation detection technology, has the advantages of high specificity, high sensitivity and mild reaction conditions by virtue of accurate target recognition and specific cutting, and has good application prospects in human pathogen and plant pathogen detection. However, the application of the technology in the detection of the Pasteurella multocida is still in a preliminary stage, the prior related research still needs to rely on complex nucleic acid extraction steps or special fluorescence detection equipment, and the technical characteristics of rapidness, simplicity and low cost of the technology cannot be fully exerted, so that the technology is not widely applied to the actual detection scene of the Pasteurella multocida. The method combines the two, not only realizes the mass amplification of nucleic acid and provides basic support for accurate identification, but also realizes the accurate cutting of targets, so that the detection is more accurate and rapid, and the cost is reduced and the efficiency is improved for the cattle raising industry. Aiming at the related technology, the existing method for detecting the Pasteurella multocida has the problems of long detection period and poor specificity or relies on large equipment and professional operation, but the detection technology based on the CRISPR/Cas12a has the defects of complex flow and insufficient field applicability, and the development of a CRISPR/Cas12a detection product and scheme which are more suitable for the actual detection requirements of animal husbandry is needed. Disclosure of Invention The invention provides a detection kit and a detection method for detecting the Pasteurella multocida based on CRISPR/Cas12a, aiming at solving the technical defects that the existing detection product of the Pasteurella multocida depends on professional equipment and personnel when in use, the detection is long in time consumption and the instant detection requirement is difficult to meet. In order to achieve the above purpose, the specific technical scheme of the invention is as follows: The invention provides a CRISPR/Cas12 a-based bovine pasteurella multocida detection kit, which comprises a isothermal amplification reagent, a CRISPR/Cas12a reagent and a nucleic acid detection test strip; the isothermal amplification system reagent comprises a primer pair for amplifying a bovine pasteurella multocida conserved gene (kmt gene), wherein an amplification product obtained by isothermal amplification of the primer pair comprises a PAM site (TTTN sequence) required by the CRISPR/Cas12a reagent recognition; The isothermal amplification system reagent is used for any one of a recombinase polymerase mediated isothermal nucleic acid amplification (RPA), RT-LAMP and HRCA isothermal amplification method; The isothermal amplification system reagent is used for a recombinase polymerase mediated isothermal nucleic acid amplification (RPA); The nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 4; the CRISPR/Cas12a reagent contains crRNA of the Pasteurella multocida kmt gene, cas12a protein and a report probe; the crRNA can specifically recognize a target sequence of an a