Multiple PCR detection reagent kit for pathogenic bacterium and its detecting method

A detection kit and pathogenic bacteria technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of lack of rapid and sensitive detection and identification technology, difficult detection and laborious detection of dairy cow mastitis pathogenic bacteria, and reach the market Wide application prospects

Inactive Publication Date: 2009-11-18
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0003] At present, my country still lacks rapid and sensitive detection and identification technology for the pathogenic bacteria of dairy cow mastitis. Conventional microbiological detection and identification methods are time-consuming and laborious, and it is difficult to detect quickly and accurately

Method used

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  • Multiple PCR detection reagent kit for pathogenic bacterium and its detecting method

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Embodiment Construction

[0035] (1) Collection of disease materials and cultivation of pathogenic bacteria

[0036] When collecting milk samples, first wash the cow’s udder with warm water and wipe it clean, then wipe the teat with 75% alcohol cotton ball or 5% iodine tincture cotton ball, squeeze out the first to third handfuls of milk in each milk area, and then squeeze the milk Put it into a pre-sterilized test tube, the amount of milk is 5mL / tube, mark it, and store it at 4°C for inspection. Shake the collected fresh milk samples, absorb 0.1mL of each milk sample and inoculate them on 10% sheep blood plate culture medium, place them in a 37°C incubator for 48 hours, observe the colony shape, pick individual colonies with different shapes and inoculate them in sheep Blood agar medium, cultured at 37°C for 48h. The purified cultured colonies were picked and inoculated in THB liquid medium for 18 hours with vigorous shaking at 37°C.

[0037] (2) Preparation of pathogenic bacteria template DNA (boil...

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Abstract

The present invention aims at the 16S-23S rRNA interval of the main pathogenic bacteria Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli of dairy cow mastitis, synthesizes 4 pairs of corresponding specific amplification primers, and applies a multiplex PCR reaction system to the cow udder detection of pathogenic bacteria. The lengths of the amplified fragments were 162bp for Streptococcus agalactiae, 264bp for Streptococcus dysgalactiae, 439bp for Staphylococcus aureus and 544bp for Escherichia coli. The multiplex PCR detection kit and detection method have strong specificity and sensitivity. It can detect and identify four kinds of pathogenic bacteria at the same time, so as to achieve the purpose of reducing the workload and cost of PCR diagnosis.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a multiplex PCR detection kit and a detection method for four pathogenic bacteria of cow mastitis. Background technique [0002] Bovine mastitis (Bovine Mastitis) is the inflammation of the mammary gland tissue of dairy cows, which is mainly caused by the infection of pathogenic microorganisms in the mammary gland tissue. It is a common and frequently-occurring disease of dairy cows. There are about 150 kinds of pathogenic bacteria causing mastitis in cows, mainly Staphylococcus aureus, Streptococcus and Escherichia coli, accounting for more than 90% of the whole cow mastitis cases. According to clinical manifestations, it can be divided into recessive and clinical mastitis. The incidence of clinical mastitis in dairy cows in my country is about 33.4%, resulting in decreased milk production, discarded sick milk, and severe cases of pus, gangrene, and atroph...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 崔玉东朱战波朴范泽
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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