Kit for identifying experimental animal cattle and sheep brucella and detection method thereof

Abstract

The invention relates to the technical field of bacterial detection and specifically relates to a kit for identifying experimental animal cattle and sheep brucella and a detection method thereof. Thekit comprises PCR reaction liquor, a standard positive template and a negative quality control standard substance, wherein the PCR reaction liquor comprises two pairs of primers for identifying cattleand sheep brucella: upstream brucella melitensis: 5'-CGC TGT CAC TGT TGC AAG TAT G-3'; downstream brucella melitensis: 5'-TCT CGC ATG CGC TAT GAT CT-3'; upstream bovine brucella: 5'-TTG AAG TCT GGC GAG CAT GA-3'; downstream bovine brucella: 5'-TCT CGC ATG CGC TAT GAT CT-3'; the standard positive template comprises a cattle standard positive template and a sheep standard positive template; the cattle standard positive template contains a pMD19-T recombinant plasmid composed of nucleotide fragments with 113 basic groups containing bovine brucella highly conserved genes; the sheep standard positive template contains the pMD19-T recombinant plasmid composed of nucleotide fragments with 128 basic groups containing brucella melitensis highly conserved genes; the two recombinant plasmids are amplified in escherichia coli DH5alpha competent cells.

Description

technical field [0001] The invention relates to the technical field of bacterial detection, in particular to a kit for differentiating animal cattle and sheep Brucella for experiments and a detection method thereof. Background technique [0002] Brucellosis is one of the most common zoonotic diseases in the world. It is called a potential bioterror weapon by the World Health Organization and can cause huge economic losses. At the same time, cattle and sheep brucellosis are also commonly used experimental animals in the teaching of scientific research fields such as animal husbandry and veterinary medicine. The detection of experimental animal diseases will improve the quality of experimental animals, thereby improving the accuracy of experimental results. In addition, avoid carrying or infection Animals with zoonotic diseases are used as experimental animals to cause biosafety problems. The most prevalent in China is Brucella ovis, three types of Brucella bovine and porcine...

AI Technical Summary

Problems solved by technology

Among the above methods, bacterial culture is time-consuming, easy to pollute the surrounding environment, and there is a risk of laboratory personnel infection. The cultivation of Brucella is required to be carried out in a biosafety level 3 laboratory. Bacterial culture methods are rarely used at present.

There are many kinds of serological detection methods, which are the standard method for epidemiological surveillance of brucellosis, but due to the cross-reactivity of Brucella species and other Gram-negative bacteria, the detection results have false positives, especially in the small intestine. Yersinia coli O:9

In summary, the isolation and identification of pathogenic sources by conventional methods is demanding, time-consuming, labor-intensive, high-risk, and low-reliability of test results

Although fluorescent quantitative PCR has high specificity and short time, it requires high experimental conditions and technical level, and it is extremely inconvenient for practical application.

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