Method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology

A technology of Enterobacter sakazakii and labeled fluorescence, which is applied in the field of microbial detection and can solve the problems of complex operation, high detection cost, and cumbersome operation

Active Publication Date: 2012-10-17
广州生凌医疗科技有限公司
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AI Technical Summary

Problems solved by technology

However, the traditional detection methods have disadvantages such as cumbersome operation, long time consumption, and easy missed detection.
Molecular detection methods include conventional PCR methods, but conventional PCR requires electrophoresis after amplification, which is complicated to operate and easily causes pollution
However, the probe method PCR has the disadvantages of high detection cost and short shelf life of reagents.

Method used

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  • Method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology
  • Method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology
  • Method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology

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Embodiment 1

[0064] Detection of Enterobacter sakazakii in milk powder using non-labeled fluorescent PCR combined with HRM analysis technology of the present invention

[0065] DNA extraction

[0066] Weigh 25 g of the sample by aseptic operation, add it to 225 mL of nutrient broth for cultivation, and incubate at 36±1°C for 18 hours. Take 1.5 mL of the culture and centrifuge at 10,000 rpm for 2 min; add 500 μL of TE buffer to the precipitate, repeatedly pipette to resuspend it, add 30 μL of 10% SDS and 15 μL of proteinase K, mix well, and incubate at 37°C for 1 hour; NaCl, mix well, then add 80ul CTAB / NaCl solution, mix well and then incubate at 65°C for 10min; add an equal volume of phenol / chloroform / isoamyl alcohol and mix well, centrifuge for 4-5min, transfer the supernatant to a In a new tube, add 0.6-0.8 times the volume of isopropanol, mix gently until the DNA precipitates, and the precipitate can be centrifuged slightly; after the precipitate is washed with 1mL of 70% ethanol, cen...

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Abstract

The invention discloses a method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology. The method is characterized by comprising the following steps: A, designing a primer pair according to the OmpA gene of Enterobacter sakazakii; B, after sample DNA is extracted, carrying out non-labeled fluorescence PCR amplification by using the designed primer pair; and C, carrying out HRM typing analysis on PCR amplification products by using a fluorescence ration PCR instrument with an HRM module so as to determine the genotype of Enterobacter sakazakii. The objective of the invention is as follows: to overcome disadvantages in the prior art, the method for detecting Enterobacter sakazakii by employing non-labeled fluorescence PCR technology and HRM analysis technology with the advantages of simple and rapid operation, accurate detection results and low usage cost is provided.

Description

technical field [0001] The invention relates to a method for detecting Enterobacter sakazakii by non-labeled fluorescent PCR and high-resolution melting curve (HRM) analysis, belonging to the field of microbial detection. Background technique [0002] Enterobacter sakazakii (Enterobacter sakazakii) is a type of Enterobacteriaceae. It was renamed Enterobacter sakazakii from Enterobacter cloacae in 1980. Recently, some international scholars suggested that it be renamed Cronobacter spp. , but has not been officially confirmed. Enterobacter sakazakii can cause severe neonatal meningitis, enterocolitis and bacteremia, and the mortality rate is as high as 50%. According to the biochemical characteristics and ribosomal spacer sequence of Enterobacter sakazakii, many scholars in the world have divided it into two branches. The representative strains are ATCC 51329 and ATCC 29544 of the American Type Culture Collection. [0003] Fluorescent PCR detection technology can be roughly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/01
Inventor 蔡先全张宪臣柏建山朱兴全邱德义伍朝晖简志华杨洁雅
Owner 广州生凌医疗科技有限公司
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