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Primer and probe for specific detection on enterobacter sakazakii and applications of primer and probe

An Enterobacter sakazakii, specific technology, applied in the field of microbial detection, can solve the problems of insufficient rigor and accuracy, large medium raw materials, difficult to determine results, etc., to save laboratory space, ensure accuracy, and avoid false negatives. Effect

Inactive Publication Date: 2015-11-25
张贵海
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The disadvantages of the above medium screening method include: 1. The total detection time takes about 5 days, and a large number of different culture dishes need to be inoculated, which is time-consuming and laborious; 2. A large amount of medium raw materials and consumables are required; 3. A large amount of space is required for placement These dishes, and the labor-intensive cleaning of the dishes, do not allow for rapid and accurate detection
[0023] 1. Low specificity: due to the design of primers and probes, the detection of opportunistic pathogenic bacteria in food is almost limited to the detection of one pathogenic bacteria, and the simultaneous detection of one-step experiments is rarely realized, and it cannot completely include All species of Enterobacter zirconium and Enterobacter;
[0024] 2. High false positive rate: dead Enterobacter sakazakii is harmless, but can still be detected
Due to the accumulation of dead cells showing a high background of inactivated Enterobacteriaceae cells, as well as the formation of aerosols of free DNA fragments during the experimental operation will lead to false positives in the experimental results, which will increase by continuing the follow-up experiments workload;
[0025] 3. High false negative rate: the false negative phenomenon due to the presence of PCR inhibitors cannot be ruled out;
[0026] 4. The result is difficult to judge: it is not rigorous enough to judge the result simply by the Ct value
[0027] It can be seen that the existing conventional detection methods have their disadvantages for the detection of Enterobacter sakazakii, and it is urgent to develop a method that can quickly and accurately detect Enterobacter sakazakii

Method used

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  • Primer and probe for specific detection on enterobacter sakazakii and applications of primer and probe
  • Primer and probe for specific detection on enterobacter sakazakii and applications of primer and probe
  • Primer and probe for specific detection on enterobacter sakazakii and applications of primer and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] In this example, specific kit products are used to detect Enterobacter sakazakii in food, especially in infant formula milk powder. The kit product is based on the method of real-time quantitative PCR and includes the following reagents:

[0095] Reagent S: ethidium bromide monoazide bromine aqueous solution 125 μg / mL;

[0096] Reagent L: DNA extraction solution, namely 0.1% Chelex100 (chelating resin) aqueous solution, can extract the DNA of Gram-negative bacteria (such as Enterobacteriaceae) within 30 minutes, and the extracted DNA can be directly used as a DNA template for PCR ;

[0097] Reagent 1: Contains specific primers and probes, the final concentration of the primers is 0.4 μmol / L, the final concentration of the probes is 0.2 μmol / L, MgCl 2 , dNTPs, PCR reaction buffer;

[0098] The sequence structure of the primers is as follows:

[0099] Enterobacter sakazakii-F: 5′-AGGGGATATTGTCCCCTGAAACAG-3′;

[0100] Enterobacter sakazakii-R: 5′-AAACGAGAATAAGCCGCGCAT...

Embodiment 2

[0138] In this example, product A is used to detect Enterobacter sakazakii and other Enterobacteriaceae bacteria in food, especially in infant formula milk powder. A product is based on the method of real-time quantitative PCR, including the following reagents:

[0139] Reagent S: ethidium bromide monoazide bromine aqueous solution 125 μg / mL;

[0140] Reagent L: DNA extraction solution, namely 0.1% Chelex100 (chelating resin) aqueous solution, can extract the DNA of Gram-negative bacteria (such as Enterobacteriaceae) within 30 minutes, and the extracted DNA can be directly used as a DNA template for PCR ;

[0141] Reagent 1: Contains specific primers and probes, the final concentration of the primers is 0.4 μmol / L, the final concentration of the probes is 0.2 μmol / L, MgCl 2 , dNTPs, PCR reaction buffer;

[0142] The sequence structure of the primers is as follows:

[0143] Enterobacter sakazakii-F: 5′-AGGGGATATTGTCCCCTGAAACAG-3′;

[0144] Enterobacter sakazakii-R: 5′-AAAC...

Embodiment 3

[0186] The method for the simultaneous specific detection of Enterobacteriaceae bacteria and Enterobacter sakazakii described in this example uses the kit described in Example 2, specifically comprising:

[0187] (1) Extraction of DNA (using reagent S and reagent L)

[0188] Take 100 g (mL) of the test sample and dissolve it in an Erlenmeyer flask filled with 900 mL of buffered peptone water preheated to 37 °C, and incubate at 37 °C for 18 hours for the first enrichment. Pipette 450 μl of buffered peptone water into a 1.5 ml centrifuge tube. Add 50 μl of the first enriched sample and mix well. Incubate at 37°C for 3-4h for secondary enrichment.

[0189] Pipette 300 μl of Reagent S (ethidium bromide monoazide bromide (EMA): 125 μg / mL) into a new 1.5ml centrifuge tube, then add 100 μl of the secondary enriched sample, and then incubate in the dark room for 5 minutes, then incubate under 500W light Lower exposure for 5min. Centrifuge at 8,000 g for 5 min and remove the supern...

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Abstract

The invention belongs to the technical field of microbiological detection, in particular relates to a primer and a probe for specific detection on enterobacter sakazakii and applications of the primer and the probe, and further discloses a method for specific detection on enterobacter sakazakii. The primer for specific detection on enterobacter sakazakii has the following sequential structure: enterobacter sakazakii-F: 5'-AGG GGA TAT TGT CCCC TG AAA CAG-3'; enterobacter sakazakii-R: 5'-AAA CGA GAA TAA GCC GCG CAT T-3'; the internal positive control-F: 5'-TGT GAA ATA CCG CAC AGA TG-3'; and the internal positive control (IAC)-R:5'-AGC TGG CGT AAT AGC GAA G-3. According to the primer and the probe for specific detection on enterobacter sakazakii, the specific design is carried out aiming at the characteristics of enterobacter sakazakii, and thus the accuracy of the result is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, in particular to a primer, a probe and an application thereof for specifically detecting Enterobacter sakazakii, and further discloses a method for specifically detecting Enterobacter sakazakii. Background technique [0002] Enterobacter sakazakii, belonging to the family Enterobacteriaceae, is a gram-negative bacterium parasitic in the intestines of humans and animals. It has no spores, perinatal flagella, and can move. It includes Escherichia, Salmonella, and Shigella. Genus, Klebsiella, Serratia, Proteus, Yersinia and other genera. Enterobacter sakazakii can cause meningitis, sepsis and necrotizing colitis in infants and premature infants, with a mortality rate as high as 50%, which has attracted the attention of relevant departments in many countries around the world. The National Foodborne Disease Surveillance Network has listed Enterobacter sakazakii as a key surveillance p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q2521/531C12Q2545/101C12Q2563/173Y02A50/30
Inventor 张贵海崔溢王亚卿
Owner 张贵海
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