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Method for detecting cronobacter sakazakii as well as kit and primer thereof

A kit and bacillus technology, applied in the field of microbial detection, can solve the problems of cumbersome operation and time-consuming detection methods, and achieve the effects of high detection efficiency, reliable detection results, and simple result judgment

Inactive Publication Date: 2013-08-14
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a new method for detecting Cronobacter sakazakii and its kit in order to overcome the defects of the existing detection methods for Cronobacter sakazakii, such as time-consuming and cumbersome operations. and primers

Method used

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  • Method for detecting cronobacter sakazakii as well as kit and primer thereof
  • Method for detecting cronobacter sakazakii as well as kit and primer thereof
  • Method for detecting cronobacter sakazakii as well as kit and primer thereof

Examples

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Embodiment 1

[0027] Example 1 Detection of Cronobacter sakazakii strains

[0028] (1) The standard strain ATCC29544 of the genus Cronobacter sakazakii was detected by PCR using the primer pair and detection method of the present invention.

[0029] The sequences of the primer pairs used are as follows:

[0030] SEN2-L: 5'-ACTGGCTTGGGGCTAATA-3' (SEQ ID NO: 1),

[0031] SEN2-R: 5'-AGAGGCGGATAAATCTTGT-3' (SEQ ID NO: 2).

[0032] Using the above primer pairs, using the genome DNA of the standard strain of Cronobacter sakazakii ATCC29544 as a template, the PCR reaction system and reaction program were established and optimized, and through single factor, multifactor test and hybridization test, a suitable for Detection of the reaction system and reaction parameters of Cronobacter sakazakii strain.

[0033] The PCR reaction system is: 1×PCR reaction buffer, 10-15mmol / L Mg 2+ , 0.2-0.3mmol / L dNTP, 0.1-0.3μM primer SEN2-L, 0.1-0.3μM primer SEN2-R, Taq enzyme 0.05-0.1U / μL, DNA template 10-100ng...

Embodiment 2

[0046] Detection of Artificial Contamination of Cronobacter sakazakii Standard Strain ATCC29544

[0047] Preparation of artificially contaminated samples: After sterilizing 100mL of 10% skim milk solution, take dilutions of 10 -7 , 10 -8 and 10 -9Add 1mL each of the pure culture solution of Cronobacter sakazakii ATCC29544 (440cfu, 44cfu and 4.4cfu) to the above-mentioned sterilized skim milk solution. Place on a shaker, culture at 37°C, 180r / min, sample every 2h, and co-cultivate for 10h. Boiling method (Chen, W., S. Yu, C. Zhang, J. Zhang, C. Shi, Y. Hu, B. Suo, H. Cao, and X. Shi, 2011, Development of a single base extension- tag microarray for the detection of pathogenic Vibrio species in seafood. Applied microbiology and biotechnology 89 (6): 1979-1990.) Genomic DNA was extracted, and PCR reaction was carried out using the aforementioned optimal reaction system and optimal amplification program. The result is as Figure 4 shown by Figure 4 It can be clearly seen tha...

Embodiment 3

[0049] The 30 food samples (including powdered milk and vegetables) were purchased from farmers' markets and large supermarkets in Shanghai. Add 50g sample to 450mL buffer peptone water (buffer peptone water, BPW, pH8.0) enrichment medium for enrichment culture, after enrichment at 37°C for 18h, take 1mL of each sample into a 1.5mL centrifuge tube, and use Boiling method (Chen, W., S. Yu, C. Zhang, J. Zhang, C. Shi, Y. Hu, B. Suo, H. Cao, and X. Shi, 2011, Development of a single base extension- tag microarray for the detection of pathogenic Vibrio species in seafood. Applied microbiology and biotechnology 89 (6): 1979-1990.) Genomic DNA was extracted, centrifuged at 12,000 rpm / min for 5 min, and 2.5 μL of the supernatant was taken as a PCR template, and sterile Water was used as a negative control, and the aforementioned optimal reaction system and optimal amplification program were used for PCR detection. Each experiment was repeated 3 times. At the same time, 18-24 hours ...

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Abstract

The invention discloses a method for detecting cronobacter sakazakii as well as a kit and a primer thereof. The method comprises the following steps of (1) extracting a genome DNA (deoxyribonucleic acid) of a sample to be detected; (2) taking the genome DNA obtained in the step (1) as a template, carrying out PCR (polymerase chain reaction) on the primers shown in sequences SEQ ID NO.1 and SEQ ID NO.2; and (3) detecting existence of a 498bp single amplification product in the reaction product obtained in the step (2), wherein the primers comprise the primers shown in the sequences SEQ ID NO.1 and SEQ ID NO.2. The kit comprises the primers. The method disclosed by the invention is short in detection time, low in cost, reliable in detection result, and simple to judge the result, and has single specificity; the method for simply, rapidly and sensitively detecting the cronobacter sakazakii is provided for the technical field of food safety inspection; and the method has significance on food safety in China.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular to a method for detecting Cronobacter sakazakii and its kit and primers. Background technique [0002] Cronobacter sakazakii (Cronobacter sakazakii) belongs to the genus Cronobacter (formerly Enterobacter sakazakii), which is a Gram-negative non-bacillus bacterium parasitic in the intestinal tract of humans and animals. Clinical studies have found that Cronobacter sakazakii is the most common opportunistic pathogen in clinical infection cases of the genus Cronobacter sakazakii, and the strain of Cronobacter sakazakii can cause fatal infections in infants, with a mortality rate as high as 40%-80% . It often results in severe clinical symptoms in infants, such as brain abscess, meningitis, necrotizing enterocolitis, and systemic sepsis. There have been reports of Cronobacter sakazakii isolates in powdered infant formula. Cronobacter sakazakii strains are important opportunist...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q2531/113C12Q2545/101
Inventor 陈万义任婧杭锋穆海菠艾连中郭本恒
Owner BRIGHT DAIRY & FOOD
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