Method for rapidly detecting bacillus coagulans and multiplex PCR reagent kit

A technology of bacillus coagulans and a kit, which is applied in the detection field of bacillus, can solve the problems of expensive instruments, difficulty in ensuring the accuracy of the detection method, and high non-specificity, and achieve the goal of reducing the economic loss of users, fast and convenient detection, and strong specificity Effect

Active Publication Date: 2016-01-20
TONGWEI
View PDF8 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, methylotrophic Bacillus and Bacillus amyloliquefaciens, which have a high molecular sequence similarity to Bacillus coagulans, have not been detected by the above methods, and it is difficult to guarantee the accuracy of the detection method
In addition, fluorescent quantitative PCR and loop-mediated isothermal amplification technology have defects such as expensive instruments, high personnel requirements or difficult quantification, and high non-specificity.

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 rapidly detecting bacillus coagulans and multiplex PCR reagent kit
  • Method for rapidly detecting bacillus coagulans and multiplex PCR reagent kit
  • Method for rapidly detecting bacillus coagulans and multiplex PCR reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Design and effect of primers for PCR amplification

[0029] 1. Experimental method

[0030] 1. PCR amplification primer design and synthesis

[0031] According to the coaG gene sequence and comK gene sequence of the Bacillus coagulans genome in GeneBank, two pairs of primers were designed with PrimerPremier5.0 software, as shown in Table 1. Primer pair 1 is coag-F and coag-R, amplifies the forward and reverse sequences of the coaG gene respectively, and is the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2; primer pair 2 is comK-F and comK-R, The forward and reverse sequences of the comK gene were respectively amplified, which were the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. The above primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0032] Table 1PCR amplification primer information

[0033]

[0034] 2. Template preparation

[0035] According to the requirements of aseptic operation, the standard strain of Baci...

Embodiment 2

[0072] Primer Specificity Experiment

[0073] 1. Experimental method

[0074] 1. DNA template preparation

[0075] According to the requirements of aseptic operation, all kinds of bacteria were cultured and DNA was extracted. details as follows:

[0076] a. Standard strain of Bacillus coagulans (CICC20138), Bacillus amyloliquefaciens and methylotrophic Bacillus: Inoculate into Bacillus coagulans liquid medium, culture at constant temperature at 37°C for 36 hours, take 2 mL of bacteria to extract DNA, and prepare DAN template ;

[0077] b. Staphylococcus, Bacillus subtilis and Bacillus licheniformis: Inoculate into LB liquid medium, culture at 37°C with constant temperature shaking (180r / min) for 24h, take 2mL of bacteria to extract DNA, and prepare DAN template;

[0078] c. Enterococcus faecalis, Lactobacillus acidophilus and Lactobacillus salivarius: Inoculate into MRS liquid medium, culture at constant temperature at 37°C for 24 hours, take 2mL of bacteria and extract DN...

Embodiment 3

[0103] Primer Sensitivity Experiment

[0104] 1. Experimental method

[0105] 1. DNA template preparation

[0106] According to the requirements of aseptic operation, the standard strain of Bacillus coagulans (CICC20138) was inoculated in the Bacillus coagulans culture liquid medium, kept at a constant temperature of 37°C for 36 hours, and 2 mL of the bacterial solution was taken to extract DNA. Bacterial genomic DNA extraction steps are as follows:

[0107] (1) Take 4 mL of the cultured bacterial liquid, centrifuge at 10,000 r / min for 1 min, and absorb the supernatant as much as possible.

[0108] (2) Add 200 μL of buffer solution to the cell pellet (weigh 20 mg of lysozyme and dissolve it in 1 mL of LTE buffer solution to make a lysozyme solution with a final concentration of 20 mg / mL, filter it through a 0.22 μm micropore and store it for future use), 37 ℃ for 40 minutes.

[0109] (3) Add 20 μL of Proteinase K solution to the tube and mix well.

[0110] (4) Add 220 μL ...

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 relates to the detection technology of bacillus and provides a method for rapidly detecting bacillus coagulans and a multiplex PCR reagent kit. The method is rapid and accurate in detection, high in sensitivity, easy to operate and low in cost. The reagent kit and method can specifically amplify coag genes and comK gene sequences in a bacillus coagulan genome, cannot obtain other bacterial gene segments through amplification and is high in specificity. Particularly for bacillus methylotrophicus and bacillus amyloliquefaciens with the high-similar molecular sequences as the bacillus coagulans, gene segments cannot be obtained through amplification. Whether bacillus coagulans exist in a sample to be detected can be accurately and effectively detected.

Description

technical field [0001] The invention relates to a detection technique for bacillus, in particular to a method for rapidly detecting bacillus coagulans and a multiplex PCR kit. Background technique [0002] Bacillus coagulans is a Gram-positive bacterium with a rod-shaped body and terminal spores that can ferment sugars to produce L-lactic acid, so it is also called "spore-forming lactic acid bacteria". Due to the safety, effectiveness, economy and environmental friendliness of the strain itself, it has become a new type of microbial feed additive approved for use in the "Catalogue of Feed Additives" (2008) issued by the Ministry of Agriculture of my country. Bacillus coagulans not only has the probiotic and health care functions of ordinary lactic acid bacteria, but also has the advantages of strong gastric acid resistance, high temperature and high pressure resistance, easy cultivation and storage, etc. It is a rising star in the field of probiotics and is widely used in me...

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): C12Q1/68
CPCC12Q1/686C12Q1/689C12Q2537/143C12Q2531/113
Inventor 苟小兰黄丹苏艳秋刘梅罗国强
Owner TONGWEI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap