Multiplex PCR method for rapid detection and identification of five Listeria species

A Listeria monocytogenes, rapid technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of laborious, low experimental repeatability, time-consuming, etc., and achieve improved sensitivity and high reliability. Reproducible, easy-to-operate results

Inactive Publication Date: 2011-12-21
INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method is time-consuming, laborious, and the experiment repeatability is relatively low, but its accuracy makes many countries still use the traditional method when setting standards
The identification of bacteria by molecular biology methods is fast and simple, but there is still room for improvement in terms of accuracy and specificity

Method used

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  • Multiplex PCR method for rapid detection and identification of five Listeria species
  • Multiplex PCR method for rapid detection and identification of five Listeria species
  • Multiplex PCR method for rapid detection and identification of five Listeria species

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Experimental program
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Effect test

Embodiment 1

[0029] Specific primer and standard strain identification experiments

[0030] 1), primer:

[0031] According to the five kinds of Liste bacteria (Single Listella, Crimeline Liszt bacteria, Implus Listor, Berlist bacteria, Ge's Listel) IAP gene sequenceIn the area, as well as related literature, use the Primer5.0 software to design the primer; select 5 upstream primers (MONOA, INO2, WELD, GRA1, Ivaa) and 1 downstream primers (LIS1B) as pre -experiments as the best combination. The primer sequences are as follows: as follows:

[0032] Monoa: 5-CAACTGCTAACACAGCTACT-3;

[0033] Upstream primer INO2: 5'-ActagcactCCAGTTTTTTAAC-3 ';

[0034] Upstream primers WELD: 5'-ACAGTAATACAACTGCACCAA-3 ';

[0035]Upstream primer GRA1: 5'-aATCAAGTAGTCTTAGCCCTTTTG-3 ';

[0036] Upstream primer IVAA: 5'-tagcagcacacgcaagcgcgcaa-3 ';

[0037] Downstream primers LIS1B: 5'-TTATACGCGACCGAAGCCAAC-3 '

[0038] The primers are synthesized by Dalian Baobi Biological Engineering Co., Ltd.; the size of the expec...

Embodiment 2

[0054] Frozen pork sample cultivation (biochemical identification as a single -increase Liszt -positive) testing experiment

[0055] 1) Preparation of sample DNA template:

[0056] The bacterial culture liquid of colonies is 1 ml, placed in a sterile 1.5 ml Eppendorf centrifuge tube, 12000 RPM centrifugal 1 min, go to clear, wash with sterilized distilled water twice, collect the bacteria; add 400 μL cracking solution (40 mmol / L Tris-acetic acid, 20 mmol / L sodium, 1 mmol / L edta, 1%SDS, pH7.8) mix, place it at 37 ° C water bath 1 h; then add 200 μL 5 mol / L.Sodium chloride solution, mixed after mixing 15 min at 13000 RPM.Take the liquid liquid and pick it twice with a phenol, pick it once with the chloroform; add twice the volume of water-free ethanol, 1 / 10 volume potassium acetate (3 mol / L, pH8.0).13000 RPM centrifugal 15 min, abandon the liquid, washed with 70%ethanol twice; after the room temperature was dry, it was dissolved in a 50 μl TE solution and settled at 4 ° C for later ...

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Abstract

The invention discloses a method for multiplex polymerase chain reaction (PCR) fast detection and five-Listeria-bacterium identification, which comprises the following steps of: 1, firstly selecting five forward primers and one reverse primer; 2, taking positive control standard bacterium liquid and bacterium liquid to be detected for being respectively prepared into genome deoxyribonucleic acid (DNA) templates; 3, respectively adding the forward primers and the reverse primer into the genome DNA templates prepared by the positive control standard bacterium liquid and the genome DNA templates prepared by the bacterium liquid to be detected for carrying out PCR amplification reaction; 4, taking amplified products for carrying out electrophoresis; and 5, judging the results. The method provided by the invention belongs to the fast, accurate and sensitive method for the multiplex PCR detection and the five-Listeria-bacterium identification, and the selection is provided for the identification of the Listeria bacterium by a molecular biological method. The efficiency is improved, the time is saved, and in addition, the detection cost is saved.

Description

Technical field [0001] The present invention involves five ways to detect and identify five ways to detect and identify five PCR. Background technique [0002] There are currently 6 types of Lisne bacteria recognized internationally: monocyte cell hyperplasia Liszt bacteria ( Histeria Monocytogenes , Referred to as Singuysi Liszto), Crusades Listol ( Listeria Ivanovii ). (Listeria Innocua ), Wil Liste bacteria ( Listeria Welshimeri ), Ge's Liszt bacteria ( Listeria Grayi ) And Sillzt bacteria ( Listeria Seeligcri To.Among them, Liste bacteria is a pathogenic bacteria that are commonly affected by humans and animals, which can cause severe infectious diseases, such as sepsis, encephalitis, and meningitis.The bacteria exist in nature, and meat, eggs, poultry, seafood, dairy products, vegetables, etc. have been proven to be the source of the infection of Listera.Safety is threatened. The bacteria can still grow and reproduce in an environment of 4 ° C. It is one of the main pathogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 郭书林陈信忠龚艳清孔繁德杨俊萍陈垚徐淑菲叶妍妍
Owner INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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