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

Method and primer for detecting lactobacilli in food

A technology for lactic acid bacteria and food, which is applied in the detection of edible lactic acid bacteria and the field of primer pairs, can solve the problems of complex components of lactic acid bacteria products, high cost of DNA sequencing, and long time-consuming, etc., to achieve easy promotion and implementation, low false positive rate, and ensure correctness sexual effect

Inactive Publication Date: 2013-04-03
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
View PDF1 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because even if the strains are preserved and cultured, it is usually difficult to distinguish
The most effective detection method is DNA sequencing. However, due to the complex composition of lactic acid bacteria products, which are often a mixture of various lactic acid bacteria, the interference of direct sequencing is serious; it takes a long time to isolate and culture the strains before sequencing.
What's more serious is that the cost of DNA sequencing is high, and the cost of daily use by manufacturers and food supervision agencies is too high to be easily implemented. Even if a corresponding method is formulated, it is difficult to implement it on a large scale, making this method useless

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 and primer for detecting lactobacilli in food
  • Method and primer for detecting lactobacilli in food
  • Method and primer for detecting lactobacilli in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Including the extraction of the bacterial genome of lactic acid bacteria in the food of embodiment 1

[0033] There are 24 imported and exported yogurt samples that can be purchased from the market, including 11 imported products and 13 domestic or joint venture brands. Tiangen Bacterial DNA Extraction Kit (Product No. DP302-02) was used to extract the whole genome of bacteria in 24 yogurt products. DNA. That is, take 1ml of yogurt liquid from different samples, centrifuge at 10,000rpm for 1 minute, discard the supernatant, and then extract the precipitate according to the manufacturer's instructions of the kit to obtain the bacterial genome extract. Determine the DNA concentration by measuring the OD260 / 280 ratio of the extract.

Embodiment 2

[0034] The PCR detection of embodiment 2 lactic acid bacteria strains

[0035] According to the inventor's research on the safety and sequencing of strains from various manufacturers in the early stage, the merger primer pairs shown in the following table 1 were designed and commissioned by Shanghai Yingjun Biotechnology Co., Ltd. The extracted bacterial genome extracts were subjected to PCR amplification, and the genome extracts of Escherichia coli and Enterobacter sakazakii (also known as Enterobacter sakazakii) were used as controls.

[0036] Table 1

[0037]

[0038]

[0039] Note: In Table 1, W represents equimolar amounts of A and T, Y represents equimolar amounts of T and C, R represents equimolar amounts of A and G; the sequence of primer A2 is shown in SEQ ID NO.1, primer The sequence of A3 is shown in SEQ ID NO.2, the sequence of primer A4 is shown in SEQ ID NO.3, the sequence of primer A5 is shown in SEQ ID NO.4, and the sequence of primer S2 is shown in SEQ ID...

Embodiment 3

[0049] Embodiment 3PCR detects the sequencing verification of the result of lactic acid bacteria strain

[0050] Each batch of the above-mentioned 24 products whose strains were stated to be stable by the manufacturer was tested as in Example 2, 99.2% showed positive results, and 0.8% showed negative results. For further verification, the PCR amplification products used in the positive and negative results were sampled separately for conventional 16S rRNA gene sequencing (using Illumina genome analyzer using 100bppaired-end mode for high-throughput sequencing), and there were no positive results. There was 1 false positive result and 25% of negative results were false negative results. Due to the low false positive rate of the method of the present invention, more than 99% of the workload requiring sequencing can be eliminated even if the sequencing is superimposed.

[0051]

[0052]

[0053]

[0054]

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 lactobacilli in food, which comprises the following steps: (1) extracting the genome DNA (deoxyribonucleic acid) of bacteria in food; (2) performing PCR (polymerase chain reaction) amplification, wherein the used primer pair is a degenerate primer pair; (3) performing electrophoresis detection on the PCR amplification product, wherein if a unique PCR amplification band is detected, the detection result is positive, otherwise the detection result is negative; if the detection result is positive, step (4) does not need to be performed; and if the detection result is negative, the step (4) needs to be performed; and (4) optionally sequencing the PCR amplification product. The invention also provides a primer and the like used in the method. According to the method disclosed by the invention, products of various manufacturers, containing single or multiple lactobacilli, can be directly detected, and the detection result can effectively eliminate false positive, thereby ensuring that the method is convenient to popularize and implement; and sequencing operation can be further performed on the product of which the detection result is negative, thereby ensuring the correctness of the detection result.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, in particular, the invention relates to a method for detection of edible lactic acid bacteria and the pair of primers used therein. Background technique [0002] Lactic acid bacteria are a group of Gram-positive bacteria with different morphology, metabolic properties and physiological characteristics. Because it has many beneficial metabolic characteristics, it is widely used in many industries such as light industry, food, medicine and feed industry. At present, the lactic acid bacteria found in nature are divided into 23 genera in bacterial taxonomy. The strains commonly used in microecological preparations include Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Leuconostoc, Pediococcus and Bacillus licheniformis etc. [0003] Most lactic acid bacteria are generally considered safe, so the corresponding detection standards are not perfect, so the detection of l...

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): C12Q1/68C12Q1/14C12Q1/04C12N15/11C12R1/23C12R1/46C12R1/245C12R1/225C12R1/01
CPCC12Q1/04C12Q1/14C12Q1/686C12Q2531/113
Inventor 杨捷琳倪胜王敏吕蓉杨柳潘良文
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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