Cronobacter sakazakii strain detecting method, kit and primer

A technology of Cronobacter and a kit, which is applied in the field of microbial detection, can solve the problems of long detection time and achieve the effects of short detection time, simple result judgment, and improved detection efficiency

Active Publication Date: 2013-03-20
BRIGHT DAIRY & FOOD CO LTD
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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 overcome the long detection time defect of the existing traditional technology, and provide a PCR detection method, nucleic acid and primer pair for Cronobacter bacterial strains.

Method used

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  • Cronobacter sakazakii strain detecting method, kit and primer
  • Cronobacter sakazakii strain detecting method, kit and primer
  • Cronobacter sakazakii strain detecting method, kit and primer

Examples

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Effect test

Embodiment 1

[0026] PCR detection method of Cronobacter strains

[0027] Step 1, primer synthesis

[0028] Synthesize primers capable of PCR amplifying the conserved sequence in the Cronobacter gyrB gene sequence (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.), the primer sequence is as follows:

[0029] SEN2-L: 5'-ATGGATAAAGAGGGCTACAG-3' (SEQ ID NO: 1),

[0030] SEN2-R: 5'-CGCCTGATTCTTACGGTTAC-3' (SEQ ID NO: 2).

[0031] Step 2, the system and reaction parameters of the PCR detection method

[0032] Using the above primers, using the genomic DNA of the standard strain of Cronobacter genus as a template, the PCR reaction system and reaction procedures were established and optimized. After single factor, multifactor experiments and hybridization experiments, it was found that the following reaction systems and reaction procedures can be A single amplification product of about 438bp was obtained. The PCR reaction system is: 1×PCR reaction buffer, 10-15mmol / L ...

Embodiment 2

[0045] Detection of artificial contamination of Cronobacter sakazakii standard strain ATCC25944

[0046] Preparation of artificially contaminated samples: Mix 10mL of milk with 89mL of liquid TSB medium, add 1mL of diluted Kronobacter sakazakii ATCC2594 pure culture solution (7CFU / mL), at 37°C, 150r / min Under culture for 12h. Samples were taken every 2 hours, genomic DNA was extracted by water boiling, and PCR detection was carried out according to the optimized conditions. The PCR detection method for Cronobacter strains established in Example 1 was used for PCR detection, and the bacteria could be detected 8 hours after artificial contamination.

[0047]

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Abstract

The invention discloses a Cronobacter sakazakii strain detecting method and kit. The method comprises the following steps of: (1) extracting genome DNA (Deoxyribose Nucleic Acid) of a sample to be detected and carrying out PCR (Polymerase Chain Reaction) amplification by taking the genome DNA as a template and a Cronobacter sakazakii specific amplification primer pair as a primers, wherein one primer in the primer pair has the sequence shown in SEQ ID NO.1, and the other primer has the sequence shown in SEQ ID NO.2; and (2) detecting the existence of a single amplification product at a 438bp position. The method provided by the invention is short in Cronobacter sakazakii strain detecting time, capable of reducing the detecting cost and increasing the detecting efficiency, single in specificity, reliable in detection result and simple in result judgment. The invention provides a simple, rapid and sensitive Cronobacter sakazakii strain detecting method for the technical field of food safety detection, which has greater 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 strains, a kit and primers thereof. Background technique [0002] Cronobacter spp is a Gram-negative non-spore-forming bacterium that parasitizes the intestinal tract of humans and animals. As an important foodborne opportunistic pathogen, Cronobacter strains can cause fatal infections in infants, with a mortality rate as high as 10%-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 strains are important opportunistic pathogens that endanger the health of infants and young children through formula powder. Newborn or premature infants are at risk of contracting Cronobacter strains through consumption of infant formula contaminated with Cronobacter ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 陈万义艾连中任婧穆海菠杭锋郭本恒李云飞
Owner BRIGHT DAIRY & FOOD CO LTD
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