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Loop-mediated isothermal amplification detection primer group, detection method and detection kit for enterobacter sakazakii

An Enterobacter sakazakii, ring-mediated isothermal technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of complicated operation, difficult to popularize and use, etc. Short detection time, rapid detection and the effect of ensuring the safety of milk powder

Active Publication Date: 2011-08-17
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

With the development of molecular biology technology, molecular detection methods such as PCR and fluorescence quantitative PCR technology are gradually applied to the rapid detection of Enterobacter sakazakii. Disadvantages such as promotion and use
At present, there is still a lack of a relatively systematic rapid detection method

Method used

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  • Loop-mediated isothermal amplification detection primer group, detection method and detection kit for enterobacter sakazakii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Sample pretreatment

[0033] Dissolve 1g of a laboratory proficiency testing comparison sample (freeze-dried milk powder) in 1mL of buffered peptone water (purchased from Guangdong Huankai Microbial Technology Co., Ltd.), then add 9ml of 44°C preheated buffered peptone water, fully shake to dissolve , incubate at 36°C±1°C for 6 hours, transfer 1mL to 10mL modified lauryl sulfate tryptone broth (mLST-Vm) (purchased from Guangdong Huankai Microbial Technology Co., Ltd.), and incubate at 44°C±0.5°C for 20h, Obtain bacterial cultures.

[0034] 2. Bacterial DNA extraction

[0035] Take 1mL of bacterial culture and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μL TE solution to suspend and mix well, put in 100℃ water bath for 10min, ice bath for 3min, centrifuge at 12000r / min for 5min, take the supernatant for later use , the supernatant contains bacterial DNA, which serves as template DNA.

[0036] 3. LAMP amplification

[003...

Embodiment 2

[0045] 1. Sample pretreatment

[0046] Add 100g of a milk powder sample (1) in the market to 900ml 44°C preheated buffered peptone water (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.), fully stir and dissolve, incubate at 36°C±1°C for 6h, absorb 1mL and transfer to 10ml Modified lauryl sulfate tryptone broth (mLST-Vm) (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.), cultured at 44°C±0.5°C for 18±2h to obtain bacterial cultures.

[0047] Following 2,3,4 steps are identical with embodiment 1

[0048] After adding the chromogen, observe the color change directly with the naked eye, the color of the reaction tube turns orange, and Enterobacter sakazakii is negative, indicating that Enterobacter sakazakii was not detected in the sample.

[0049] 5. Confirmation of Enterobacter sakazakii chromogenic medium

[0050] A certain milk powder sample (1) in step 1 was cultured in buffered peptone water and modified lauryl sulfate tryptone broth,...

Embodiment 3

[0053] 1. Sample pretreatment

[0054] Add 100g of a milk powder sample (2) in the market to 900ml 44°C preheated buffered peptone water (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.), fully stir and dissolve, incubate at 36°C±1°C for 6h, absorb 1mL and transfer to 10ml Modified lauryl sulfate tryptone broth (mLST-Vm) (purchased from Guangdong Huankai Microbiology Technology Co., Ltd.), cultured at 44°C±0.5°C for 18±2h to obtain bacterial cultures.

[0055] Following 2,3,4 steps are identical with embodiment 1

[0056] After adding the chromogen, observe the color change directly with the naked eye, the color of the reaction tube turns green, and Enterobacter sakazakii is positive, indicating that Enterobacter sakazakii was detected in the sample.

[0057] 5. Chromogenic biochemical confirmation of Enterobacter sakazakii

[0058] The bacterial culture obtained after a certain milk powder sample (2) in step 1 was cultured step by step with buffered pepto...

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Abstract

The invention discloses a loop-mediated isothermal amplification detection primer group, a detection method and a detection kit for enterobacter sakazakii. Aiming at ompA genes of the enterobacter sakazakii, in the invention, a specific detection primer group, a detection kit containing the detection primer group, and a detection method utilizing the detection kit are designed and selected, so that detection is carried out on samples to be detected, especially milk powder, thus determining whether the specific gene segments of the enterobacter sakazakii exist, and further determining whether the samples to be detected contains the enterobacter sakazakii. According to the invention, the detection kit and the detection method have the advantages of high sensitivity, strong specificity, short detection time, without a PCR (polymerase chain reaction) instrument and an electrophoresis apparatus, are simple for operation process, are especially suitable for a basic detection mechanism and food enterprises for self check, and have important meaning for fast detecting milk powder and ensuring the safety of the milk powder.

Description

Technical field: [0001] The present invention relates to a rapid detection technology for pathogenic bacteria based on loop-mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) technology, in particular to detection primers specific to a fragment of the specific gene ompA of Enterobacter sakazakii A detection method and a detection kit for detecting Enterobacter sakazakii by using the detection primer set using the loop-mediated isothermal amplification method LAMP. Background technique: [0002] Enterobacter sakazakii (Enterobacter sakazakii) is a Gram-negative non-bacillus bacterium belonging to the Enterobacteriaceae genus. In 1961, Franklin et al first reported two cases of infant meningitis caused by Enterobacter sakazakii, and then a series of cases of Enterobacter sakazakii infection in newborns were reported successively around the world. Enterobacter sakazakii can cause severe neonatal meningitis, enterocolitis, and bacteremia, and can ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCY02A50/30
Inventor 徐晓可吴清平张淑红张菊梅
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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