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A detection primer and detection kit for Cryptobacter pyogenes

A technology of Cryptobacter pyogenes and a detection kit, which is applied in the field of molecular biology, can solve the problems of laboratories without high cleanliness, reduce the independent space of aerosol, and high reagent costs, and achieve clear target bands and short time-consuming , the effect of high sensitivity

Active Publication Date: 2021-04-06
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the PCR method, the advantages of using the LAMP method for detection are obvious. It does not require an expensive PCR machine, high detection sensitivity, strong specificity, and visualized results. The disadvantages of using the LAMP method for detection are also obvious. High temperature, closed space to prevent pollution, high cost of reagents, many operating steps, etc.
Grass-roots veterinary stations and other institutions are currently equipped with PCR machines, electrophoresis machines and other equipment, but they are basically not equipped with high-cleanliness laboratories or independent spaces to reduce aerosol generation

Method used

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  • A detection primer and detection kit for Cryptobacter pyogenes
  • A detection primer and detection kit for Cryptobacter pyogenes
  • A detection primer and detection kit for Cryptobacter pyogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Preparation of DNA template

[0047] ①Liquid culture: Take 1 mL of bacterial liquid and centrifuge at 12,000×g for 1 min, discard the supernatant, add 100 μL of deionized water to suspend the bacteria, bathe in boiling water for 10 min, and after cooling, centrifuge at 12,000×g for 10 min, and take the supernatant as a template.

[0048] ②Solid culture: Scrape the pure culture of C. pyogenes, suspend it in 100 μL of deionized water, bathe in boiling water for 10 minutes, and centrifuge at 12000×g for 10 minutes after cooling, and take the supernatant as a template.

[0049] ③Lung tissue, lymph node tissue, pus: take samples and add PBS to grind, incubate at room temperature for 10min, 5000r / min, take 500μL supernatant, add 50μL 10% SDS solution and 10μL 20μg / mL proteinase K, bathe in 56℃ water for 2h, add 500μL Tris to saturate Phenol, after mixing well, centrifuge at 12000×g / min for 10min, take the supernatant, add an equal amount of chloroform:isoamyl alcohol (24:1...

Embodiment 2

[0073] Use the F1 / R1 primer pair with good specificity in Example 1 to detect clinical samples, including tissues, pus, and the like.

[0074] 1. DNA preparation

[0075] Take the sample and grind it with PBS, incubate at room temperature for 10min, 5000r / min, take 500μL of supernatant, add 50μL of 10% SDS solution and 10μL of 20μg / mL proteinase K, bathe in water at 56℃ for 2h, add 500μL of Tris saturated phenol, mix thoroughly, 12000×g Centrifuge at 12000×g / min for 10 min, take the supernatant, add an equal amount of chloroform:isoamyl alcohol (24:1), mix thoroughly, and centrifuge at 12000×g / min for 10 min, take the supernatant and add 2 times the volume of absolute ethanol, -20 Stand at ℃ for 20 minutes, centrifuge at 12000×g / min for 10 minutes, discard the supernatant, add 70% ethanol to wash the precipitate, discard the ethanol, add 20 μL sterilized deionized water to dissolve the precipitate, and store at -20°C for PCR detection.

[0076] Add 500 μL of deionized water t...

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Abstract

The invention belongs to the field of molecular biology, and in particular relates to a detection primer and a detection kit for Cryptobacterium pyogenes. The invention establishes a specific and sensitive PCR method for the hemolysin PLO gene sequence of the Cryptobacterium pyogenes, can accurately detect the Cryptobacterium pyogenes, and distinguish other pathogens of goat purulent infection. The invention provides positive control bacteria and negative control bacteria for users to determine the quality of the kit and evaluate the accuracy of the method. The kit and method provided by the present invention can be used in basic-level routine animal disease diagnosis laboratories, the target bands are clear and easy to distinguish, and the PCR reaction can be completed within 1 hour. are of great significance.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a detection primer and a detection kit for Cryptobacterium pyogenes. Background technique [0002] Trueperella pyogenes (Trueperella pyogenes) is a Gram-positive short rod-shaped bacterium, which is an opportunistic pathogenic bacterium of important economic animals such as cattle, sheep and pigs. And the purulent infection of mucous membrane, causes bigger economic loss to aquaculture. In most cases, C. pyogenes infection causes chronic wasting disease; in severe cases, death is often caused by sepsis. In addition, C. pyogenes can cause infections in various animals such as antelopes, camels, dogs, cats, elephants, gazelles, saberbucks, parrots, horses, deer, reindeer, and turkeys. [0003] Cryptobacter pyogenes is often co-infected with other pathogenic bacteria. In this laboratory, bacteria were isolated and identified from goat lymphadenitis and other suppurati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 张素辉沈克飞杨柳徐登峰许国洋付利芝
Owner CHONGQING ACAD OF ANIMAL SCI
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