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Burkholderia gladioli LAMP (loop-mediated isothermal amplification) constant-temperature rapid detection method

A technology of Holderia puberkia and Holderia, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of long time-consuming, cumbersome detection steps, detection Low efficiency and other problems, to achieve good stability, high efficiency, fast and easy detection, and high sensitivity

Active Publication Date: 2022-03-15
GUANGZHOU INST FOR FOOD INSPECTION(GUANGZHOU INSPECTION CENT FOR WINE & SPIRITS)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, traditional physiological, biochemical and immunological methods are mainly used for the detection of Burkholderia gladiolus (Pseudomonas cocos subspecies). Purification, biochemical identification, serum identification, toxicological test, the detection operation steps are cumbersome, time-consuming, and detection efficiency is low. In major food safety guarantee tasks or sudden food poisoning incidents, traditional detection methods are difficult to meet the requirements for food safety. Provide timely and accurate judgment requirements

Method used

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  • Burkholderia gladioli LAMP (loop-mediated isothermal amplification) constant-temperature rapid detection method
  • Burkholderia gladioli LAMP (loop-mediated isothermal amplification) constant-temperature rapid detection method
  • Burkholderia gladioli LAMP (loop-mediated isothermal amplification) constant-temperature rapid detection method

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Experimental program
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Effect test

Embodiment 1

[0032] The rapid detection method of Burkholderia gladiolus LAMP constant temperature amplification comprises the following steps:

[0033] Step 1: Prepare the DNA sample to be tested and the specific primers for LAMP constant temperature amplification of the DNA sample to be tested for LAMP constant temperature amplification;

[0034] In this step, according to the requirements of the bacterial genomic DNA extraction kit (the kit is produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., product number is DP302), the sample is taken to extract the DNA sample to be tested, and the DNA sample to be tested is subjected to ultra-micro After spectrophotometer measurement, its A260 / 280 is between 1.7-2.0, the DNA concentration is >10ng / μL, and the DNA solution that meets the requirements is stored at -20°C for future use.

[0035]The specific primers for constant temperature amplification of the LAMP are obtained by designing and synthesizing specific gene sequences of Burk...

Embodiment 2

[0053] This embodiment is to detect the specificity of the LAMP constant temperature amplification reaction system and LAMP constant temperature amplification primers of the present invention, specifically: use the LAMP constant temperature amplification reaction system of Example 1 to test 33 standard bacterial strains in this embodiment The DNA sample is detected, and the fluorescent signal of the amplified product is detected. See the test results figure 2 and Table 3.

[0054] from figure 2 As can be seen from Table 3, the present invention is positive to Burkholderia gladiolus DNA sample amplification, Burkholderia gladiolus (CICC10574) standard bacterial strain and Burkholderia gladiolus ( Wild strain) (confirmed as Burkholderia gladiolus) had an obvious S-type amplification curve, while the amplification results for other standard strains were all negative, and there was no obvious S-type amplification curve.

[0055] Conclusion: The detection method of the present...

Embodiment 3

[0059] This embodiment detects the sensitivity of the LAMP constant temperature amplification reaction system of the present invention and the LAMP constant temperature amplification specific primers, specifically: the concentration of the extracted Burkholderia gladiolus DNA is determined, and its concentration is determined to be 150.0ng / μL, the DNA was serially diluted 10 times to obtain 15.0ng / μL, 1.5ng / μL, 150.0pg / μL, 15.0pg / μL, 1.5pg / μL, 0.15pg / μL, according to the LAMP of Example 1 Constant temperature amplification reaction system and conditions LAMP constant temperature amplification was carried out, and the experiment was repeated 6 times.

[0060] Results: The concentrations of LAMP fluorescently amplified DNA of Burkholderia gladioli were significantly higher at 150.0ng / μL, 15.0ng / μL, 1.5ng / μL, 150.0pg / μL, 15.0pg / μL, and 1.5pg / μL. The S-type amplification curve shows that the sensitivity of this method is 1.5pg / μL of DNA, and the amplification map is shown in ima...

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Abstract

The invention provides a burkholderia gladioli LAMP (loop-mediated isothermal amplification) constant-temperature rapid detection method. The method comprises the following steps: 1, preparing a DNA (deoxyribonucleic acid) sample to be detected and LAMP constant-temperature amplification specific primers; wherein the LAMP isothermal amplification specific primer is obtained by selecting a burkholderia gladioli specific gene sequence for synthesis, and the gene adopts a burkholderia gladioli (pseudomonas arenata subsp. Oryzae) hydrolase (cayF) gene; the LAMP isothermal amplification specific primers comprise an outer primer F3, an outer primer B3, an inner primer FIB, an inner primer BIP, a loop primer LF and a loop primer LB; and 2, carrying out LAMP isothermal amplification on the to-be-detected DNA sample by adopting the LAMP isothermal amplification reaction system, and detecting a fluorescence signal of an amplification product to obtain a detection result. The Burkholderia gladioli detection kit has good specificity and high sensitivity on Burkholderia gladioli, and can be used for rapid detection of Burkholderia gladioli in food.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a constant-temperature amplification method for detecting Burkholderia gladiolus LAMP in food. Background technique [0002] Pseudomona cocovenenans (Pseudomona cocovenenans) is a new food poisoning bacterium discovered in Northeast fermented rice noodle poisoning food in 1977, and was classified as Burkholderia gladioli in 2003. ) is a pathogenic prototype. Cereal fermented products (fermented corn flour, glutinous corn glutinous rice ball powder, corn starch, fermented glutinous millet, vinegar jelly, etc.), spoiled white fungus, and potato products (potato vermicelli, sweet potato starch, yam starch, etc.) are easily contaminated by this bacteria. Burkholderia gladiolus can produce toxin and oryzinic acid, which can cause highly pathogenic food poisoning. Food poisoning incidents caused by the oryzae acid toxin produced by this bacteria often occur in s...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2531/119
Inventor 肖剑梁美丹宋安华孙雪奇戚平雷燕曹霞飞胡雯艳黄铁城叶春嫦
Owner GUANGZHOU INST FOR FOOD INSPECTION(GUANGZHOU INSPECTION CENT FOR WINE & SPIRITS)
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