Specific detection molecular markers 3283 and 3316 for Yersinia enterocolitica and rapid detection method for Yersinia enterocolitica

A technology for enterocolitis and Yersinia enterocolitis, which is applied in the field of gene detection of Yersinia enterocolitica, can solve the problems of difficulty in timely detection of pollution, high time and labor costs, and long separation and identification cycle. Achieve the effect of reducing the reliability of results, reducing time and economic costs, and eliminating false positive results

Active Publication Date: 2020-03-27
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This has just caused the isolation and identification period of Yersinia enterocolitica to be often very long (7-10 days), and detection rate is low
High time and labor costs for Y. enterocolitica detection make timely detection of Y. enterocolitica contamination in food difficult

Method used

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  • Specific detection molecular markers 3283 and 3316 for Yersinia enterocolitica and rapid detection method for Yersinia enterocolitica
  • Specific detection molecular markers 3283 and 3316 for Yersinia enterocolitica and rapid detection method for Yersinia enterocolitica
  • Specific detection molecular markers 3283 and 3316 for Yersinia enterocolitica and rapid detection method for Yersinia enterocolitica

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Search for Yersinia enterocolitica Species-Specific Molecular Tags

[0036] The screening of Yersinia enterocolitica species-specific molecular signatures was mainly based on pan-genome analysis results. A total of 16 strains of Yersinia enterocolitica complete genome data (from the NCBI database) and 23 strains of Yersinia enterocolitica complete genome draft data were selected (the strains were preserved in this experiment and included in this experiment. The whole genome data of 78 strains of other Yersinia were analyzed by pan-genome analysis (for Yersinia except Yersinia enterocolitica species, see Table 1 for the species and quantity). The pan-genome analysis adopts the MP method in the prokaryotic pan-genome analysis pipeline (Pan-Genomics Analysis Pipeline, PGAP), and the analysis results are processed through the local Perl script to obtain the core gene and non-core gene information of all strains. Then, the specific genes of Yersinia enterocolitica...

Embodiment 2

[0039] Example 2 Establishment of a rapid detection method for molecular targets of Yersinia enterocolitica

[0040] Design specific amplification primers according to the sequence molecular tags 3283 (SEQ ID NO: 1) and 3316 (SEQ ID NO: 2), the primer sequences are shown in Table 2:

[0041] Table 2: Index 3283 and 3316 primer sequences

[0042]

[0043]

[0044] The genomic DNA of 23 strains of Yersinia enterocolitica isolated from food in our laboratory was used as a template for PCR amplification verification.

[0045] The PCR system is 25 μL, including 2.5 μL of 10×PCR reaction buffer, 25 mmol / L MgCl 2 2.0 μL, 0.5 μL of 10 mmol / L (each) dNTPs, 0.5 μL of 10 μmol / L primer, 1 U of Taq enzyme, 0.5-1 μL of DNA template, and sterilized double distilled water to make up the volume to 25 μL.

[0046] The PCR reaction conditions for the 3283 molecular tag are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, exte...

Embodiment 3

[0052] Example 3 Specificity evaluation results of Yersinia enterocolitica PCR detection method

[0053] Take 37 Yersinia strains of non-enterocolitis Yersinia such as Yersinia intermediate, Yersinia freundii, Yersinia ruckeri, Yersinia burgdorferi, and 32 strains of cereus Bacillus, Pseudomonas aeruginosa and other non-Yersinia strains were tested for the specificity of Yersinia enterocolitica molecular signature 3283 and 3316. The genomic DNA of the above corresponding bacterial strains was extracted as a template for PCR amplification according to the PCR amplification system and amplification conditions described in Example 1, and finally detected by agarose gel electrophoresis. The detection results are shown in Table 4 and Table 5 (the corresponding electrophoresis result pictures are respectively image 3 , 4 and Figure 5 , 6 ).

[0054] Table 4: Specificity test results of molecular tags 3283 and 3316 in 32 strains of Bacillus cereus, Pseudomonas aeruginosa and o...

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Abstract

The invention discloses two detection targets 3283/3316 for Yersinia enterocolitica and detection primers for the Yersinia enterocolitica. Sequences of the detection targets are shown in SEQ ID NO: 1and SEQ ID NO: 2. The detection targets for the Yersinia enterocolitica, disclosed by the invention, can be applied to rapid detection on the Yersinia enterocolitica in samples of foods and the like.The special detection primers disclosed by the invention particularly comprise a 3283 marker forward primer 3283-F, a 3283 marker reverse primer 3283-R, a 3316 marker forward primer 3316-F and a 3316marker reverse primer 3316-R. Through PCR amplification and agarose gel electrophoresis detection, a detection result can be obtained from an electrophoresis product result. The specific detection molecular markers and the rapid detection method can be used for effectively detecting the Yersinia enterocolitica in the samples of the foods and the like, have the advantages of rapidness, high efficiency, simplicity in operation and the like and can be applied to the fields of foods, inspection and quarantine and the like.

Description

technical field [0001] The invention belongs to the technical field of gene detection of Yersinia enterocolitica, and specifically relates to two species-specific detection molecular tags 3283 and 3316 of Yersinia enterocolitica and a rapid detection method thereof. Background technique [0002] Nucleic acid amplification test (NAAT) is a rapid, easy-to-use, sensitive, economical and low-labor-cost detection method for the detection of pathogenic bacteria in food and clinical environments, which has gradually emerged in recent years. Gastrointestinal multiplex panel (GIMP) designed based on this method is also more and more used in clinical testing. [0003] Yersinia enterocolitica (Yersinia enterocolitica, Ye) is a Gram-negative pathogenic bacteria that widely exists in the world and can cause Yersinia disease in humans and animals. Handling or eating undercooked pork is a leading cause of illness in humans. Patients with Yersinia usually present with a clinical syndrome ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/10C12N15/11
CPCC12Q1/689
Inventor 吴清平刘鸣王涓丁郁张菊梅叶青华陈谋通薛亮吴诗雷涛庞锐吴浩明
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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