Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously

A detection method and technology for killing Pasteurella multocida, applied in the field of PCR detection, can solve the problems of cumbersome process, time-consuming, low detection rate, etc., and achieve the effects of improving detection efficiency, shortening detection time, and reducing requirements

Inactive Publication Date: 2017-03-22
HEBEI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional diagnostic techniques such as separation and culture, immunological tests, etc. are time-consuming and laborious, and are not suitable for rapid clinical diagnosis and large-scale epidemiological investigation. Traditional methods have defects such as cumbersome process, time-consuming, low detection rate and missed detection.

Method used

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  • Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously
  • Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously
  • Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Optimization of the annealing temperature for the five-fold PCR system of Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae

[0025] (1) Sample pretreatment

[0026] Standard Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae were respectively cultured in the corresponding medium or culture solution, among which Staphylococcus aureus and Pseudomonas aeruginosa were cultured for 24 hours , Pasteurella multocida and Bordetella bronchiseptica were cultured for 36 hours, and Mycoplasma pneumoniae were cultured for 7 days, and the corresponding DNA samples were extracted from the bacterial liquid or colonies.

[0027] (2) Bacterial DNA extraction

[0028] The present invention adopts bacterial genomic DNA extraction kit to extract DNA, and the steps are as follows:

[0029] ① Dissolve the above-mentioned 5 kinds of pathoge...

Embodiment 2

[0041] Example 2 Detection of five-fold PCR artificial samples of Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae

[0042] (1) Sample pretreatment

[0043] Sample feces and tracheal secretions from the animal to be tested.

[0044] (2) Bacterial DNA extraction

[0045] Same as Example 1

[0046] (3) Establishment of multiplex PCR reaction system

[0047] PCR reaction system is 50 μl, Mix 25 μl, primer 7 μl, upstream and downstream primers for Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae are 1 μl, 1 μl, 0.5 μl, 0.5 μl respectively , 0.5 μl, DNA was positive control 5 μl, negative control 0 μl, sample feces and tracheal secretions were 2 μl each, and the remaining sterile water was added to 50 μl. Reaction conditions: pre-denaturation at 94°C for 5 minutes; cycle at 94°C for 30s, 60°C for 30s, and 72°C for 30s, a total of 30 c...

Embodiment 3

[0050] Example 3 Detection of five-fold PCR samples of Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae

[0051] (1) Sample pretreatment

[0052] Sample feces and tracheal secretions from the animal to be tested.

[0053] (2) Bacterial DNA extraction

[0054] Same as Example 1

[0055] (3) Establishment of multiplex PCR reaction system

[0056] PCR reaction system is 50 μl, Mix 25 μl, primer 7 μl, upstream and downstream primers for Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae are 1 μl, 1 μl, 0.5 μl, 0.5 μl respectively , 0.5 μl, DNA was positive control 5 μl, negative control 0 μl, sample feces and tracheal secretions were 2 μl each, and the remaining sterile water was added to 50 μl. Reaction conditions: pre-denaturation at 94°C for 5 minutes; cycle at 94°C for 30s, 60°C for 30s, and 72°C for 30s, a total of 30 cycles; fina...

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Abstract

The invention discloses a quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously. According to the method, firstly, genes with conservative sequences of staphylococcus aureus, pseudomonas aeruginosa, pasteurella multocida, bordetella bronchiseptica and mycoplasma pulmonis are screened out and taken as targets for PCR detection, specific primers of corresponding conservative sequences are synthesized and amplified respectively, five pairs of primers are placed in one PCR system, and the quintuple PCR detection method capable of detecting five pathogens once from a sample directly is established through optimization of each item. Compared with a conventional PCR detection method, the detection method has the characteristics of rapidness, sensitivity, simplicity, convenience and accuracy, and the requirements for detection people and the detection cost are reduced.

Description

technical field [0001] The invention relates to a PCR detection method in the field of biotechnology, in particular to a five-fold PCR detection method capable of simultaneously detecting Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae . Background technique [0002] The existing national standard "Experimental Animal Microbiological Grades and Monitoring" GB 14922.2-2011 detects pathogens - mainly traditional isolation and culture, biochemical identification, serotype analysis and special identification experiments. In clinical practice, Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella multocida, Bordetella bronchiseptica and Mycoplasma pneumoniae are common and important pathogens in experimental animals, and they are prone to mixed infections. Traditional diagnostic techniques such as separation and culture, immunological tests, etc. are time-consuming and laborious, and are not suitable ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12Q1/14
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 王俊霞郑龙祝岩波尤红煜连伟光刘健敏张东明
Owner HEBEI MEDICAL UNIVERSITY
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