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

Method for detecting viable bacteria in staphylococcus aureus

A staphylococcus and lysostaphin technology, applied in the field of microbial detection, can solve problems such as inability to effectively distinguish live bacteria from dead bacteria, high detection values, and inaccurate detection results, so as to avoid false positive results and achieve high extraction efficiency , the effect of improving sensitivity

Inactive Publication Date: 2018-09-07
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF3 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PMA-qPCR or EMA-qPCR detection technology cannot effectively distinguish live bacteria from dead bacteria when detecting live bacteria treated at low temperature, and it is prone to high detection values ​​and inaccurate detection results

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
  • Method for detecting viable bacteria in staphylococcus aureus
  • Method for detecting viable bacteria in staphylococcus aureus
  • Method for detecting viable bacteria in staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0054] 3. Preparation of dead bacteria

[0055] Centrifuge the bacterial suspension at 5000 g for 10 min at 4°C, discard the supernatant, and resuspend the bacterial cells with 0.1% peptone water. The concentration of the liquid after resuspension was adjusted to 10 7 -10 8 CFU / mL, and divided into two equally. One of them was heated at 90°C for 20 minutes to prepare dead bacteria, and the other was used as live bacteria.

Embodiment 1

[0057] Primer inclusion and exclusivity verification

[0058] Using 18 strains, including 10 standard strains of Staphylococcus aureus and 8 other known strains, bacterial DNA was extracted by CTAB method. The PCR reaction system (25 μL) included 12.5 μL Master Mix, 2 μL template DNA, 1 μL each of upstream and downstream primers, the concentration of the primers was 4000 nM, and 8.5 μL of water.

[0059] The primer sequence is: upstream primer 5'-CACCTGAAACAAAGCATCCTAAA-3', downstream primer 5'-CGCTAAGCCACGTCCATATT-3';

[0060] Reaction program: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 62°C for 1 min, and 40 cycles. The amplified products were subjected to electrophoresis in 2.5% agarose gel electrophoresis prepared in 1×TE buffer.

[0061] As shown in Table 1, using the above primers, only the DNA of Staphylococcus aureus showed positive signals, and the DNA of other strains showed no positive signals. This result indicated that the ...

Embodiment 2

[0065] Optimization of SDS concentration

[0066] SDS was dissolved in 0.1% peptone water to prepare a 20% SDS stock solution, which was then sterilized.

[0067] Staphylococcus aureus (ATCC6538) bacteria liquid (10 7 CFU / mL) was heat-treated at 90°C for 35s, and centrifuged at 5000g for 10min at 4°C. Then resuspended with different concentrations of SDS, the concentrations of SDS were 0, 25, 50, 100, 250, 500, 1000ppm. Take 100 μL of the solution and spread it on a plate, incubate at 37°C for 24 hours, and observe the number of colonies. Select the optimal concentration according to the inhibition of SDS to Staphylococcus aureus live bacteria.

[0068] Such as figure 1 As shown, when the SDS concentration is 0, 25, 50, and 100 ppm, the corresponding logCFU values ​​are 6.5, 6.3, 6.2, and 6.0, respectively. When the SDS concentration was 250, 500, 1000ppm, the logCFU values ​​were 4, 4, 0, respectively. It can be seen from the figure that when the SDS concentration is 10...

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 provides a method for detecting viable bacteria in staphylococcus aureus in order to solve the problems of false positive and false negative when a PMA (Propidium Monoazide)-qPCR (quantitative Polymerase Chain Reaction) method is adopted for detecting the viable bacteria in the prior art. According to the method provided by the invention, an SDS (Sodium Dodecyl Sulfonate) treatment step is added before PMA treatment on the basis of the PMA-qPCR method, a lysostaphin treatment step is added before DNA (Deoxyribonucleic Acid) extracting, and bacillus cereus DNA is added as an amplification internal reference during a qPCR process, so that the problems of false positive and false negative in an original method are well solved, and the sensitivity and the specificity of the detecting method are improved.

Description

technical field [0001] The invention relates to a method for detecting viable staphylococcus aureus in milk, belonging to the field of microorganism detection. Background technique [0002] Staphylococcus aureus is a Gram-positive, spherical pathogen capable of producing enterotoxins. It can cause food poisoning or clinical infections such as cellulitis and osteomyelitis. Staphylococcus aureus is a major cause of bovine mastitis and human disease infections in the dairy industry. The presence of S. aureus in raw milk is also a potential source of pathogens in the dairy food chain, with the attendant risk of food contamination. Staphylococcus aureus is highly prevalent in many countries, including the United States and Italy. Therefore, an accurate method for detecting Staphylococcus aureus is very necessary in epidemiology. [0003] Conventional methods for detecting S. aureus are cheap and reliable, but laborious and time consuming. However, PCR-based methods can overc...

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 Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/14C12Q1/06C12R1/445
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 王加启郑楠董蕾刘慧敏孟璐兰图邢萌茹
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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