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

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

Inactive Publication Date: 2007-01-24
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) Collect disease materials and cultivate pathogenic bacteria

[0036] When milk samples are collected, first wash and wipe the udders of the cows with warm water, then wipe the nipples with 75% alcohol cotton balls or 5% iodine tincture cotton balls, squeeze out the first to third milk in each milk area, and then squeeze the milk Put it into a pre-sterilized test tube with a milk volume of 5 mL / tube, mark it, and store it at 4°C for inspection. Shake the collected fresh milk samples, draw 0.1 mL of each milk sample and inoculate them into 10% sheep blood plate medium, place them in a 37°C incubator for 48 hours, observe the morphology of the colonies, and pick individual colonies with different morphologies and inoculate them in sheep Blood agar medium, incubate at 37°C for 48h. Pick the purified culture colonies and inoculate them in THB liquid medium at 37°C with vigorous shaking for 18 hours.

[0037] (2) Preparation of pathogen template DNA (boiling method)

[0038] ...

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Abstract

The present invention synthesizes four pairs of specific amplifying primers separately corresponding to the 16S-23S rRNA of main pathogenic bacteria of milk cow mammitis including staphylococcus aureus, agalactia streptococcus, delactation streptococcus and colibacillus, and detects pathogenic bacteria of milk cow mammitis with multiple PCR reaction system. The amplified segments lengths are 439 bp for staphylococcus aureus, 162 bp for agalactia streptococcus, 264 bp for delactation streptococcus and 544 bp for colibacillus. The multiple PCR detection reagent kit and detection method has powerful specificity, high sensitivity, capacity of detecting and identifying four kinds of pathogenic bacteria simultaneously, simple detection process and low detection cost.

Description

Technical field [0001] The invention belongs to the field of molecular biology detection, and specifically relates to a multiple PCR detection kit for four pathogens of dairy cow mastitis and a detection method thereof. Background technique [0002] Bovine Mastitis (Bovine Mastitis) is an inflammation of the breast tissue of dairy cows, which is mainly caused by infection of pathogenic microorganisms in the breast tissue. It is a common and frequently-occurring disease in dairy cows. There are about 150 kinds of pathogenic bacteria that cause mastitis in dairy cows, mainly Staphylococcus aureus, streptococcus and E. coli, which account for more than 90% of the entire dairy 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 and waste of sick milk. In severe cases, the milk area becomes purulent, gangren...

Claims

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Application Information

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Patent Type & Authority Applications(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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