Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for detecting Cronobacter sakazakii and its kit and primers

Through specific primer pairs for PCR reaction and agarose gel electrophoresis detection, the problem of long time and error in detecting Cronobacter sakazakii was solved, rapid, low-cost specific detection was achieved, and the efficiency of food safety detection was improved. efficiency and reliability.

Active Publication Date: 2016-04-27
BRIGHT DAIRY & FOOD CO LTD
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for detecting Cronobacter sakazakii and its kit and primers
  • A method for detecting Cronobacter sakazakii and its kit and primers
  • A method for detecting Cronobacter sakazakii and its kit and primers

Examples

Experimental program
Comparison scheme
Effect test

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 (Cronobacters akazakii) 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 / LMg 2+ , 0.2-0.3mmol / LdNTP, 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 3-5mi...

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 asingle base extension-tag microarray for the detection of pathogenic Vibriospecies in seafood. Applied microbiology and biotechnology 89 (6) : 1979-1990.) Genomic DNA was extracted, and PCR reaction was carried out by 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...

Embodiment 3

[0051] The 30 food samples (including milk powder and vegetables) were purchased from farmers' markets and large supermarkets in Shanghai. Add 50g sample to 450mL buffer peptone water (bufferpeptonewater, BPW, pH8.0) enrichment medium for enrichment culture, after enrichment at 37°C for 18h, take 1mL of each sample and put it into a 1.5mL centrifuge tube, and use the boiling method (Chen, W., S. Yu, C. Zhang, J. Zhang, C. Shi, Y. Hu, B. Suo, H. Cao, and X. Shi, 2011, Development of asingle base extension-tag microarray for the detection of pathogenic Vibriospecies in seafood. Applied microbiology and biotechnology 89 (6): 1979 -1990.) Genomic DNA was extracted, centrifuged at 12,000rpm / min for 5min, and 2.5 μL of supernatant was taken as a PCR template, and sterile water was used as a negative control, and PCR was carried out using the aforementioned optimal reaction system and optimal amplification program detection. Each experiment was repeated 3 times. At the same time, a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 (Cronobacters akazakii), a kit and primers thereof. Background technique [0002] Cronobacter sakazakii (Cronobactersakazakii) belongs to the genus Cronobacter (Cronobacterspp.) (formerly known as Enterobacter sakazakii), and 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. Crono...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com