Triple PCR (Polymerase Chain Reaction) detection method for simultaneously detecting plurality of types of pathogens

A detection method and sequence technology, applied in the field of PCR detection, can solve the problems of inappropriate epidemiological investigation, time-consuming, cumbersome process, etc., and achieve the effects of rapid detection standardization, shortening detection time, and improving detection efficiency.

Inactive Publication Date: 2017-05-10
河北意和医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional diagnostic techniques such as isolation and culture, immunological tests, etc. are time-consuming and laborious, and are not suitable for rapid clinical dia

Method used

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  • Triple PCR (Polymerase Chain Reaction) detection method for simultaneously detecting plurality of types of pathogens
  • Triple PCR (Polymerase Chain Reaction) detection method for simultaneously detecting plurality of types of pathogens
  • Triple PCR (Polymerase Chain Reaction) detection method for simultaneously detecting plurality of types of pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Optimization of the annealing temperature of the triple PCR system of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae

[0028] (1) Sample pretreatment

[0029] The standard Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae were cultured in the corresponding medium or culture solution, among which Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae were cultured for 24h, and the bacteria were collected. The corresponding DNA samples were extracted from the solution or colony.

[0030] (2) Bacterial DNA extraction

[0031] The present invention adopts the bacterial genome DNA extraction kit to extract DNA, and the steps are as follows:

[0032] Dissolve the above-mentioned 3 kinds of pathogen bacteria liquids or colonies in buffer GA, shake and suspend;

[0033] Add 20 μl Proteinase K solution, mix well, add 220 μl Buffer GB, shake for 15 s, 70°C for 10 min, add 220 μl absolute ethanol, sha...

Embodiment 2

[0044] Example 2 Detection of triple PCR artificial samples of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae

[0045] (1) Sample pretreatment

[0046] Take a sample feces from the animal to be tested.

[0047] (2) Bacterial DNA extraction

[0048] Same as Example 1

[0049] (3) Establishment of multiplex PCR reaction system

[0050] The PCR reaction system is 50 μl, including: Mix 25 μl, primers 6.5 μl, of which, the universal upstream and downstream primers for Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and bacteria are 1 μl, 1 μl, 1 μl, 0.25 μl, respectively, DNA The positive control was 3 μl, the negative control was 0 μl, the sample feces were 3 μl each, and the rest of the sterile water was supplemented to 50 μl. Reaction conditions: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; final extension at 72°C for 7 min. 10 μl of PCR products were electrophoresed in 2.5% a...

Embodiment 3

[0053] Example 3 Detection of triple PCR samples of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae

[0054] (1) Sample pretreatment

[0055] Take sample feces from the live animal to be tested.

[0056] (2) Bacterial DNA extraction

[0057] Same as Example 1

[0058] (3) Establishment of multiplex PCR reaction system

[0059] The PCR reaction system is 50 μl, including: Mix 25 μl, primers 6.5 μl (1 μl, 1 μl, 1 μl, 0.25 μl of universal upstream and downstream primers for Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and bacteria, respectively), DNAs are The positive control was 3 μl, the negative control was 0 μl, the sample feces were 3 μl each, and the rest of the sterile water was supplemented to 50 μl. Reaction conditions: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; final extension at 72°C for 7 min. 10 μl of PCR products were electrophoresed in 2.5% agarose gel at 12...

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Abstract

The invention discloses a triple PCR (Polymerase Chain Reaction) detection method for simultaneously detecting a plurality of types of pathogens. The triple PCR detection method comprises the following steps: firstly, screening conservative and specific genes with staphylococcus aureus, pseudomonas aeruginosa and klebsiella pneumonia and common genes for bacteria and taking the genes as target spots for PCR detection; synthesizing and amplifying specific primers corresponding to conservative sequences respectively; simultaneously putting four pairs of primers into the same PCR reaction system; and optimizing all items to establish the triple PCR detection method capable of simultaneously detecting staphylococcus aureus, pseudomonas aeruginosa and klebsiella pneumonia from excrement of living animals. Meanwhile, common primers for the bacteria, which are designed by 16Sr RNA (Ribonucleic Acid), are added into the reaction system and are used as internal quality control; and in a pathogen detection process, whether the reaction system is complete or not can be judged according to the condition of a common primer amplification band and the quality of a template added into an evaluation system can be judged. The detection method disclosed by the invention is rapid, sensitive, simple, accurate and non-invasive, and the requirements on detection personnel 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 triple PCR detection method capable of simultaneously detecting Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. Background technique [0002] The detection methods for pathogens in the existing national standard "Grading and Monitoring of Laboratory Animal Microbiology" GB 14922.2-2011 are mainly based on traditional isolation and culture, biochemical identification, serotype analysis and special identification experiments. In clinical practice, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae are opportunistic pathogens of zoonotic disease, and are prone to mixed infection. Traditional diagnostic techniques such as isolation and culture and immunological tests are time-consuming and labor-intensive, and are not suitable for rapid clinical diagnosis and large-scale epidemiological investigations. The traditi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 郑龙祝岩波李丹丹张东明范晓飞
Owner 河北意和医学检验实验室有限公司
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