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Method, fluorescent PCR primers and probe for detecting burkholderia gladioli and bongkrekic acid toxin-producing strain

A technology of Burkholderia populatum and mycobacterial acid is applied in the field of molecular biology, which can solve the problems of long detection period and no molecular field involved, and achieves strong repeatability, strong practical value and specificity. Good results

Active Publication Date: 2021-04-13
SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, the commonly used detection method for Burkholderia gladiolus is GB 4789.29-2020 "National Standard for Food Safety Food Microbiological Examination Subspecies) Test”, this standard does not involve the molecular field, and to identify whether the strain produces toxins requires a toxin production test, and the detection cycle is long

Method used

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  • Method, fluorescent PCR primers and probe for detecting burkholderia gladioli and bongkrekic acid toxin-producing strain
  • Method, fluorescent PCR primers and probe for detecting burkholderia gladioli and bongkrekic acid toxin-producing strain
  • Method, fluorescent PCR primers and probe for detecting burkholderia gladioli and bongkrekic acid toxin-producing strain

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Embodiment 1

[0037] Detection of Burkholderia gladiolus in food.

[0038] (1) Design and synthesis of primers

[0039] Design and screening of specific primers and TaqMan probes for the detection of Burkholderia gladiolus, the sequences of which are:

[0040] PBA-F primer: 5'-GCTTCCGCTATCCAAATTACTACTTC-3' (shown in SEQ ID NO: 1),

[0041]PBA-R primer: 5'-ATGACAAATGTTCGAGTCAGTTGAC-3' (shown in SEQ ID NO: 2),

[0042] Probe PBA: 5'-FAM-ATGACAAATGTTCGAGTCAGTTGAC-BHQ-3' (shown in SEQ ID NO: 3).

[0043] (2) Sample DNA extraction

[0044] Extract DNA by boiling lysis method, take 1 mL of enrichment solution, add it to a 1.5 mL centrifuge tube, centrifuge at 12,000 g for 2 min, aspirate and discard the supernatant; add 500 μL of sterile water, mix well, centrifuge at 12,000 g for 2 min, aspirate and discard Supernatant; add 100 μL sterile water, boil in water for 10 min, centrifuge at 12,000 g for 2 min, and store the supernatant at -20°C for later detection; commercial DNA extraction kits c...

Embodiment 2

[0054] Detection of Burkholderia oryzae acid-producing acid strain of Burkholderia gladiolus

[0055] (1) Design and synthesis of primers

[0056] Design primers and probes for detection of Burkholderia gladiolus oryzae acid strains, and establish a fluorescent PCR for distinguishing Burkholderia oryzae acid-producing and non-producing Burkholderia oryzae acids system, its sequence is:

[0057] BA-F primer: 5'-CGATGATATAGCCGAGGTT-3' (shown in SEQ ID NO: 4),

[0058] BA-R primer: 5'-CAGGTTCCAGTGCCATTA-3' (shown in SEQ ID NO: 5),

[0059] Probe BA: 5'-FAM-CGATGGTCCGTATCTCCTGCTTGTGC-BHQ-3' (shown in SEQ ID NO: 6).

[0060] (2) DNA extraction

[0061] Extract DNA by boiling lysis method, take 1 mL of enrichment solution, add it to a 1.5 mL centrifuge tube, centrifuge at 12,000 g for 2 min, aspirate and discard the supernatant; add 500 μL of sterile water, mix well, centrifuge at 12,000 g for 2 min, aspirate and discard Supernatant; add 100 μL sterile water, boil in water for ...

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Abstract

The invention provides a method, fluorescent PCR primers and probe for detecting burkholderia gladioli and a bongkrekic acid toxin-producing strain. The fluorescent PCR primers and probe for detecting the burkholderia gladioli include a PBA-F primer with a sequence as shown in SEQ ID NO:1, a PBA-R primer with a sequence as shown in SEQ ID NO:2, and a PBA fluorescent probe with a sequence as shown in SEQ ID NO:3. According to the technical scheme, the burkholderia gladioli and thebongkrekic acid toxin-producing strain are subjected to real-time fluorescent PCR detection by adopting a two-step method; the blank of molecular detection is filled; the method can be used for rapidly screening the burkholderia gladioli and the bongkrekic acid toxin-producing strain in foods; and the method is good in specificity, high in repeatability and high in sensitivity, can check hidden dangers in time and has relatively high practical value.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a detection method for acid-producing strains of Burkholderia gladioli and oryzae bacterium, fluorescent PCR primers and probes for detection. Background technique [0002] Burkholderia gladiolus is a kind of Gram-negative short bacilli without spores, with an optimum growth temperature of 37°C and an optimum pH of 5-6, and is widely distributed in nature. Many Burkholderia gladiolus species are non-toxic, but some pathogenic species are pathogenic to animals or plants. The current study found that there are 4 pathogenic variants of the bacteria, which are B. gladioli pv. agaricicola, B. gladioli pv. alliicola, B. gladioli pv.gladioli and B. gladioli pv.cocovenenans, where pathogenic variants B. gladioli pv. cocovenenans produces oryzae acid toxin, the pathogenic variant exists in fermented grain products, spoiled white fungus and fungus and other fo...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2561/101Y02A50/30
Inventor 陈晶王晓雯李碧芳王远洋黄建飞张悦陈国培杨国武
Owner SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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