Unlock instant, AI-driven research and patent intelligence for your innovation.

Group of specific primers and probe for real-time fluorescence quantitative PCR detection of lactobacillus casei as well as detection kit

A real-time fluorescent quantitative, Lactobacillus casei technology, applied in the field of microbial detection, can solve the problems of complex detection, low accuracy, time-consuming, etc., and achieve good specificity.

Active Publication Date: 2015-07-01
SUZHOU BAIYUAN GENT CO LTD
View PDF1 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are many types of lactobacilli with similar phenotypes, so it is difficult to quickly and accurately distinguish them. Traditional quantitative detection methods rely on specific microbial culture media to separate and count live bacteria in food. The test process requires medium preparation, plate culture, colony counting, Biochemical identification and other steps, so the whole detection is complicated, time-consuming, and the accuracy rate is low

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
  • Group of specific primers and probe for real-time fluorescence quantitative PCR detection of lactobacillus casei as well as detection kit
  • Group of specific primers and probe for real-time fluorescence quantitative PCR detection of lactobacillus casei as well as detection kit
  • Group of specific primers and probe for real-time fluorescence quantitative PCR detection of lactobacillus casei as well as detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of Lactobacillus casei (DNA polymerase I gene) polA gene standard

[0034] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. The polA gene sequence is highly conserved, and its special functional region has abundant cysteine ​​and four unique insertion regions, which play an important role in DNA replication and repair. The polA gene detects Lactobacillus casei with high sensitivity and specificity. This part mainly uses PCR technology to amplify the Lactobacillus casei polA gene, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-LcpolA, and carry out corresponding PCR identification and sequencing identification, and finally After quantification, it is used as a standar...

Embodiment 2

[0091] Example 2 Preliminary establishment of real-time fluorescence quantitative PCR detection method for Lactobacillus casei

[0092] 1. Design and synthesis of specific primers and probes

[0093] Taking the conserved fragment sequence of the polA gene of Lactobacillus casei selected above as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using Primer express3 software, Primer Premier 5 software and Oligo 7 software, and the probes were detected by Taqman. Needle.

[0094] As the core of the present invention, a group of specific primers and probe nucleotide sequences for Lactobacillus casei real-time fluorescent PCR detection are as follows:

[0095] Upstream primer: LcpolA227F: 5'-CCGCGTGATGAGGATATTTATG-3'

[0096] Downstream primer: LcpolA313R:5'-AGACTTGCGAGCAAACTGGC-3'

[0097] Probe: LcpolA256P:5'-CGCCAAACGGCAAGTGCCAGA-3'.

[0098] The target nucleotide sequence of this primer and probe amplification is:

[0099] 5'-CCGC...

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 group of specific primers and probe for real-time fluorescence quantitative PCR detection of lactobacillus casei. An upstream primer is LcpolA227F:5'-CCGCGTGATGAGGATATTTATG-3'; a downstream primer is LcpolA313R:5'-AGACTTGCGAGCAAACTGGC-3'; the probe is LcpolA256P:5'-CGCCAAACGGCAAGTGCCAGA-3'; and the target nucleotide sequence amplified by the primers and probe is shown by SEQ ID No.2 in a sequence table. The invention also provides a corresponding quantitative PCR detection kit. By adopting the primers and probe as well as the kit provided by the invention, the content of lactobacillus casei in a sample to be detected can be quantified quickly, accurately and sensitively with good specificity.

Description

technical field [0001] The invention belongs to the technical field of microbial detection methods, and in particular relates to a set of specific primers and probes for real-time fluorescence quantitative PCR detection of Lactobacillus casei and a corresponding quantitative PCR detection kit. Background technique [0002] Probiotics are a kind of active microbial food raw materials that are beneficial to human health. At present, the commonly used probiotics include Lactobacillus acidophilus and Bifidobacterium, which together with Lactobacillus casei are called "healthy three beneficial bacteria". At present, there are not many studies on Lactobacillus casei. Lactobacillus casei can tolerate the defense mechanism of the organism, including enzymes in the mouth, low pH in gastric juice and bile acids in the small intestine, etc., so Lactobacillus casei can survive in large numbers in the intestinal tract after entering the human body, thereby playing a role It can regulate...

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): C12Q1/68C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2545/113C12Q2561/101C12Q2561/113
Inventor 车团结李亚鹏李琳常运朝史延桥夏秋花
Owner SUZHOU BAIYUAN GENT CO LTD