Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner

A technology for enterocolitis and Yersinia enterocolitica is applied in the field of rapid constant temperature detection of Yersinia enterocolitica, which can solve the problems of lack of generality and specificity of primers

Active Publication Date: 2017-02-22
上海市生物医药技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defects of primer versatility and specificity in the primer design of the existing LAMP technology, make full use of the abundant microbial genome sequen...

Method used

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  • Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner
  • Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner
  • Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-6

[0410] Embodiment 1-6 Yersinia enterocolitica constant temperature reaction system and detection method

[0411] Follow the steps (1) to (3) below for testing:

[0412] (1) Extraction of genomic DNA

[0413] The strain of Yersinia enterocolitica used for detection was obtained from the China Industrial Microbiology Culture Collection Center, numbered CICC21565. Take 1mL of bacterial culture and use Beijing Tiangen Bioengineering Company's Bacterial Nucleic Acid Extraction Kit to extract genomic DNA, DNA OD 260 / OD 280 It was 1.8, and the concentration was 364ng / μL.

[0414] (2) Using Yersinia enterocolitica genome DNA to be tested as a template, using self-prepared kits (see Table 2, Table 3) respectively, and preparing a reaction system according to the conditions described in Table 3, using small intestine The Yersinia coli specific amplification primer set is used as primers for constant temperature amplification reaction. The primers in Examples 1-6 are respectively p...

Embodiment 7

[0418] Example 7 Yersinia enterocolitica specific detection

[0419] Collect 28 strains of non-enterocolitis Yersinia (Table 4 and figure 1 1~23, 25~29 in ), compared these strains with Yersinia enterocolitica strains (Table 4 and figure 1 In 24) culture respectively, get 1mL bacterium liquid, adopt kit 1A, extract bacterial DNA, and with reference to the reaction system and condition of embodiment 1, carry out LAMP amplification respectively (primer group is A) and add chromogenic agent to observe .

[0420] The test results are shown in Table 4 and figure 1 as shown, figure 1Among them, 1-23 are Staphylococcus aureus, Staphylococcus aureus subsp. aureus, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria innoculus bacterium, Listeria evansiella, Salmonella enterica subsp. enterica, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella baumannii, Shigella flexn...

Embodiment 8

[0424] Embodiment 8 Sensitivity detection

[0425] Extract the DNA of bacteria CICC 21565 according to the method of Example 2, adopt kit IB, and add reaction system according to 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg and 5fg DNA gradient, other reaction conditions refer to Table 3 Example 1 The method was used to perform LAMP amplification (primer set is A) and add chromogenic reagent for observation respectively. Such as figure 2 As shown, 1-8 are respectively 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg and 5fg, NTC: negative control. figure 2 The reaction products treated with 50ng, 5ng, 500pg, 50pg, 5pg and 500fg are bright green, which is a positive result, and the reaction products of 50fg, 5fg treatment and negative control are orange, which is a negative result. The test results show that each reaction tube can still be detected when the minimum DNA content is 500fg (equivalent to about 100 bacteria), and the sensitivity is high.

[0426] According to the above det...

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Abstract

The invention discloses a method, a primer group and a kit for quickly detecting yersinia enterocolitica in a constant-temperature manner. The method comprises the following steps of extracting DNA (Deoxyribonucleic Acid) of a genome from a to-be-detected sample; using the DNA of the genome as a template, using a primer group capable of amplifying the specific sequence of the yersinia enterocolitica as primers, and carrying out constant-temperature amplification reaction under an enzyme reaction system; through judging whether a reaction result is positive or not, determining whether the yersinia enterocolitica exists in the to-be-detected sample or not. The detection method provided by the invention has high sensitivity and high specificity, is short in detection time, is simple in result decision, is convenient and quick to operate, is low in cost, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method, primers and kit for rapid and constant temperature detection of Yersinia enterocolitica. Background technique [0002] Yersinia enterocolitica (Yersinia enterocolitica) is widely distributed in nature and is one of the few intestinal pathogenic bacteria that can grow at refrigerated temperatures. In addition to causing gastrointestinal symptoms, it can also cause arthritis. Diseases such as erythema nodosum and mesenteric lymphitis can even cause sepsis and cause death. The clinical symptoms after infection by the bacteria are often not obvious, which is easy to cause misdiagnosis. Because the bacteria can survive in a low temperature environment, food stored in refrigerators is an important source of infection in modern society. Many countries have listed this bacteria as a routine inspection item for imported and exported food. [0003] At present, the ident...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119Y02A50/30
Inventor 李亦学韦朝春李园园李雪玲刘伟贾犇陆长德陆晓婷
Owner 上海市生物医药技术研究院
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