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PCR (Polymerase Chain Reaction) method and kit for quickly detecting Enterobacter sakazakii in baby formula

A technology of Enterobacter sakazakii and infant formula, which is applied in the fields of biochemical equipment and methods, microbe determination/inspection, and resistance to vector-borne diseases, etc.

Inactive Publication Date: 2012-10-24
陕西省产品质量监督检验所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the linear structure of its probe, its specificity needs to be further improved.

Method used

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  • PCR (Polymerase Chain Reaction) method and kit for quickly detecting Enterobacter sakazakii in baby formula
  • PCR (Polymerase Chain Reaction) method and kit for quickly detecting Enterobacter sakazakii in baby formula
  • PCR (Polymerase Chain Reaction) method and kit for quickly detecting Enterobacter sakazakii in baby formula

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Kit composition and preparation

[0084] 1. DNA extraction solution: including the following components: 10×TE (0.1M Tris-HCL, 0.1M EDTA pH8.8); 50ug / ul lysozyme; 1% SDS; 0.2ug / ul proteinase K.

[0085] 2. 10×PCR reaction solution: including 100mM KCL; 1200mM Tris-HCL; 1% Triton x-100; 15mM MgSO 4 ; 100mM (NH 4 ) 2 SO 4 ; 1 mg / mL BSA;

[0086] 3. Standard positive template: The standard positive template is composed of pGEMT-easy vector containing a 265-base nucleotide fragment of the conserved gene ompA of Enterobacter sakazakii.

[0087] 4. Specific primers and molecular beacons:

[0088] 5-ACCGGCGTTTCTCCTGTA-3

[0089] 5-TCAGCATGCCGTTGTCC-3

[0090] Fluorescence signal FAM-ctgcat-CGGGGTTTCTCCTGTATTCG-atgcag-DABCYL quenches the signal.

[0091] 5. Internal standard recombinant plasmid

[0092] The standard internal standard control is a pUC18T recombinant plasmid constructed from the artificially synthesized sequence containing the human MICA gene and the Mol...

Embodiment 2

[0096] Embodiment 2: the sensitivity test of kit

[0097] 1. Culture of Enterobacter sakazakii

[0098] Standard strains of Enterobacter sakazakii were purchased and cultured overnight in bacterial broth for later use.

[0099] 2. Preparation of Enterobacter sakazakii genomic DNA.

[0100] After the strain was cultured overnight in the bacterial broth, the bacteria were suspended in 500ul TE buffer, added with a final concentration of 50ug / ul lysozyme, and reacted at 37°C for 1 hour, then added a final concentration of 1% SDS and 0.2ug / ul proteinase K, After 1 h at 55°C, the DNA was extracted with phenol-chloroform.

[0101]3. PCR amplification conditions: PCR reaction system (25 μL): 2 μL of 10×PCR buffer, 1 μL of each primer pair and molecular beacon (10 μmol / L), 2 μL of dNTPs (10 mmol / L), TaqDNA polymerase (5 U / μL ) 0.2 μL, template DNA 2 μL, water 16.8 μL. PCR reaction conditions: 94°C for 3min; 94°C for 60s, 60°C for 60s, 40 cycles; observe the amplification curve in ...

Embodiment 3

[0102] Embodiment 3: the specificity test of kit

[0103] Salmonella schottmuelleri; Salmonella pafatyphi B; Salmonella typhimurium; Salmonella enreritidis; Shigella sonnei; Shlgella dysenteriae; Shigella boydii; Shigella flexnei; Enterobacteo yersinia); Proteus vuLgaris; Listeria monocytogenes; Escherichia coli (Eseheriehiaeoli) as a control, and Enterobacter sakazakii ATCC29544 strain; ATCC 51007 strain; ATCC 51024 strain DNA, positive control plasmid, internal standard recombinant plasmid, each take 5uL as a template for PCR reaction. At the same time, a negative control was set up.

[0104] The PCR reaction conditions were: PCR reaction system (25 μL): 2 μL of 10×PCR buffer, 1 μL of each primer pair and molecular beacon (10 μmol / L), 2 μL of dNTPs (10 mmol / L), 0.2 μL of Taq DNA polymerase (5 U / μL) μL, template DNA 2 μL, water 16.8 μL.

[0105] PCR reaction conditions: 94°C for 3min; 94°C for 60s, 60°C for 60s, 40 cycles.

[0106] Results Only Enterobacter sakazakii (En...

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Abstract

The invention provides a PCR (Polymerase Chain Reaction) method and kit for quickly detecting Enterobacter sakazakii in a baby formula. In the method, Enterobacter sakazakii is efficiently extracted from an artificially polluted baby formula, and is evaluated by comparison. The method comprises the following specific steps: amplifying an Enterobacter sakazakii gene used as the target gene, thus constructing a target recombinant plasmid as a standard substance for quantification; constructing an internal standard recombinant plasmid, adding into a reaction system, and amplifying together with the target gene; monitoring the reaction system, and eliminating the false negative; optimizing the fluorescent quantitative reaction system; determining the fluorescent quantitative method and the artificial pollution detection limit; and carrying out the actual detection. The invention has the advantages of high detection speed, good specificity, simple use steps and high repeatability, can further implement dual or multiple real-time PCR, and can complete the simultaneous detection of multiple food-borne pathogens.

Description

technical field [0001] The invention relates to a molecular beacon fluorescence quantitative PCR method for rapidly detecting Enterobacter sakazakii in formula milk powder for infants and young children and a kit thereof. Background technique [0002] Enterobacter sakazakii is a Gram-negative non-bacillus bacterium that parasitizes the intestinal tract of humans and animals and belongs to the genus Enterobacteriaceae. Because Enterobacter sakazakii is one of the normal intestinal flora and is an opportunistic pathogen, it has not been paid attention to clinically until 1961, when two doctors, Uremenyi and Frank in the United Kingdom, reported two cases caused by this bacteria for the first time. Meningitis cases, followed by Denmark, the United States, the Netherlands, Greece, Iceland, Belgium and other countries have reported a number of cases of Enterobacter sakazakii infection in newborns. [0003] In the food industry, Enterobacter Sakazakii is also a foodborne pathogen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 李雪玲赵建设陈勇张莉樊成唐欣蒋宏伟许慧敏王慧芳
Owner 陕西省产品质量监督检验所
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