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A Triple-PCR Detection Method for Rapid Differentiation of Non-pneumophilic, Pneumophilic and Pneumophilic Legionella Type I

A Legionella and pneumophilic technology, applied in the field of multiple PCR detection of Legionella, can solve the problems of uncertainty, multiple cross-reactivity, suspicious Legionella colonies cannot be finally identified, etc., achieve low cost, accurate and reliable results, The effect of simple method

Inactive Publication Date: 2016-04-13
无锡市疾病预防控制中心 +1
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Problems solved by technology

However, the traditional culture method has a blind spot when judging the isolated suspected Legionella colonies, the uncertainty of biochemical detection and the multiple cross-reactivity of serological detection, resulting in some suspicious Legionella colonies that cannot be finally identified

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  • A Triple-PCR Detection Method for Rapid Differentiation of Non-pneumophilic, Pneumophilic and Pneumophilic Legionella Type I

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Embodiment 1

[0017] Example 1 A triple PCR detection method for rapidly distinguishing non-pneumophilic, pneumophilic and pneumophilic type 1 Legionella, the steps are:

[0018] 1. Prepare a PCR template, pick a suspicious single colony to be detected and put it in 0.5mL sterilized nuclease-free deionized water to prepare a bacterial suspension, boil it at 100°C for 15min, and cool it for later use.

[0019] 2. Primer sequence:

[0020] 16S rRNA gene: upstream primer 5'-AAGATTAGCCTGCGTCCGAT-3', downstream primer 5'-GTCAACTTATCGCGTTTGCT-3', the expected length of the amplified fragment is 654bp;

[0021] danJ gene: upstream primer 5'-AGGTGGTTTTGGCGGATTTGG-3', downstream primer 5'-TGAATTCTGACTTGCCCCATG-3', the expected length of the amplified fragment is 285bp;

[0022] ORF9 gene: upstream primer 5'-CAGGATTACCGCTCATTATTG-3', downstream primer 5'-GTAATTCCCAGCCATTTACCAGATC-3', the expected length of the amplified fragment is 561bp.

[0023] 3. PCR reaction system: 1.6 μL dNTP, 2 μL each of t...

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Abstract

The invention relates to a triple PCR (Polymerase Chain Reaction) detection method for rapidly distinguishing non-LP (legionella pneumophila), LP and LP I, which is a legionella multiple PCR detection method and belongs to the field of microorganism molecule detection. According to the invention, primers of 16SrRNA, danJ and ORF9 genes which respectively aim at legionella, LP and LP I are utilized to optimize a dNTPs and primer concentration ratio and an annealing temperature so as to establish the triple PCR detection method for rapidly distinguishing the non-LP (legionella pneumophila), the LP and the LP I. The method disclosed by the invention is simple, convenient and rapid, is low in cost, has an accurate and reliable result and can rapidly and effectively judge dubious legionella strains cultured by separation.

Description

technical field [0001] A triple PCR detection method for rapidly distinguishing non-pneumophilic, pneumophilic and pneumophilic type I Legionella is a multiplex PCR detection method for Legionella and belongs to the field of microbial molecular detection. Background technique [0002] More than 90% of Legionnaires' disease is caused by Legionella pneumophila (Legionella Pneumophila, LP), of which 85% is LP1 type Legionella pneumonia, so Legionella pneumophila and LP1 type Legionella pneumophila are the focus of our monitoring. According to WS394-2012 "Hygienic Specifications for Centralized Air-Conditioning and Ventilation Systems in Public Places", Legionella pneumophila must not be detected in cooling tower water and condensate water of centralized air-conditioning and ventilation systems in public places. The standard detection method of this specification is the culture method. However, the traditional culture method has a blind spot when judging the isolated suspected L...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/686C12Q2537/143
Inventor 张琦周伟杰范晓东严洁沈波
Owner 无锡市疾病预防控制中心