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Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method

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

Active Publication Date: 2014-08-06
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by this invention is to provide a new method for detecting Cronobacter sakazakii in order to overcome the defects of the existing detection methods such as Cronobacter sakazakii (Cronobacter sakazakii) which are time-consuming and complicated to operate. and its kits and primers

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  • Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method
  • Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method
  • Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method

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Embodiment 1

[0029] Example 1 Detection of Cronobacter sakazakii strains

[0030] (1) Using the primer pair and detection method of the present invention to carry out PCR detection on the standard strain of Cronobacter sakazakii (Cronobacter sakazakii) ATCC29544.

[0031] The sequences of the primer pairs used are as follows:

[0032] SEN2-L: 5'-ACTGGCTTGGGGCTAATA-3' (SEQ ID NO: 1),

[0033] SEN2-R: 5'-AGAGGCGGATAAATCTTGT-3' (SEQ ID NO: 2).

[0034] Using the above primer pairs, using the genomic DNA of the standard strain of Cronobacter sakazakii ATCC29544 as a template, the PCR reaction system and reaction procedures were established and optimized. Reaction system and reaction parameters of Cronobacter sakazakii strain.

[0035] 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 / μL. The PCR amplification program is: 94-95°C pre-denaturation for 3-5...

Embodiment 2

[0048] Detection of Artificial Contamination of Cronobacter sakazakii Standard Strain ATCC29544

[0049] Preparation of artificially contaminated samples: After sterilizing 100mL of 10% skim milk solution, take dilutions of 10 -7 , 10 -8 and 10 -9 1 mL of the pure culture broth of Cronobacter sakazakii ATCC29544 (440 cfu, 44 cfu and 4.4 cfu) was added 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 performed using the aforementioned optimal reaction system and optimal amplification program. The result is as Figure 5 shown by Figure 5 It can be clearly seen th...

Embodiment 3

[0051] The 30 food samples (including milk powder and vegetables) were purchased from farmers' markets and large supermarkets in Shanghai. Add 50g of sample to 450mL buffer peptone water (buffer peptone water, BPW, pH8.0) enrichment medium for enrichment culture, after 18 hours of enrichment at 37°C, take 1mL of each sample and put it 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- tagmicroarray 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 PCR template. As a negative control, use the aforementioned optimal reaction system and optimal amplification program for PCR detection. Each experiment was repeated 3 times. At the...

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Abstract

The invention discloses a detecting method for Cronobacter sakazakii as well as a kit and primers used in the detecting method. The method comprises the following steps of (1) extracting a genome DNA (Deoxyribonucleic Acid) of a sample to be detected; (2) enabling primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 to be subjected to PCR (Polymerase Chain Reaction) by taking the genome DNA obtained in the step (1) as a template; and (3) detecting the existence of a single amplification product 498bp in the reaction product obtained in the step (2). The primers include the primers as shown in 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, single in specificity, reliable in detecting result and simple in result judgment; and in addition, the invention provides the simple, rapid and sensitive detecting method for Cronobacter sakazakii in the technical field of food safety detection, and the detecting method has important significance for 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, a kit and primers thereof. Background technique [0002] Cronobacter sakazakii, belonging to the genus Cronobacter spp. (formerly Enterobacter sakazakii), 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 fatality 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 opportunistic pathogens that en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
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 CO LTD
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