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Rapid qualitative and quantitative detection kit, detection method and application of Lactobacillus casei added in feed

A technology for the quantitative detection of Lactobacillus casei, which is applied in the field of animal husbandry and veterinary medicine, can solve the problems that the qualitative and quantitative detection methods are not scientific and fast enough, limit the development of probiotic preparations, consume a lot of time and resources, and achieve low detection standards and high sensitivity. High and accurate effect

Active Publication Date: 2018-05-25
HUBEI HAOHUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, probiotic preparations continue to develop, but the qualitative and quantitative detection methods are not scientific and fast enough, which limits the development of probiotic preparations to a certain extent.
Usually, the traditional methods of physiological and biochemical reactions and selective medium pure culture isolation and counting are very easy to limit the detection efficiency and unstable detection results due to differences in bacterial species. In addition, traditional methods will also consume a lot of time and effort. resource

Method used

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  • Rapid qualitative and quantitative detection kit, detection method and application of Lactobacillus casei added in feed
  • Rapid qualitative and quantitative detection kit, detection method and application of Lactobacillus casei added in feed
  • Rapid qualitative and quantitative detection kit, detection method and application of Lactobacillus casei added in feed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Selection of target detection genes of Lactobacillus casei:

[0027] The present invention is effective for Lactobacillus casei and other probiotics such as Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus bulgaricus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium, etc. A large number of comparisons and analyzes were performed on the genome sequences of Lactobacillus casei, and the target gene for qualitative and quantitative detection was selected. The target gene is a conserved housekeeping gene in the genome of Lactobacillus casei, and it is a single-copy gene. The qualitative and quantitative detection of Lactobacillus casei in the feed can be carried out with specific primers, and the obtained data can truly reflect the quantity of Lactobacillus casei added in the feed. The primers designed for the target gene are: 5'-TGCGGCGGTAAAGGTTG-3', 5'-CGTCTGTGTAGAAACTGCGAATG-3'; these primers can be used for qualitati...

Embodiment 2

[0029] Rapid qualitative and quantitative detection kit for Lactobacillus casei added in feed, including primers: 5'-TGCGGCGGTAAAGGTTG-3', 5'-CGTCTGTGTAGAAACTGCGAATG-3'.

[0030] Example: 3:

[0031] The detection method for the rapid qualitative and quantitative detection kit of Lactobacillus casei added in feed, including the method for pretreatment of feed samples, the method includes:

[0032] Mix 25g of the fully pulverized and homogeneous feed with 225mL of sterilized physiological saline, shake at 4°C for 1-2h at a speed of 100 rpm, and prepare a 10% uniform dilution. Aseptically perform 10-fold incremental dilution on the homogeneous dilution prepared in the previous step, select more than 2 to 3 appropriate dilutions, and extract bacterial genomic DNA or RNA as needed.

[0033] The remaining steps utilize the primers in Example 1 to carry out qualitative and quantitative identification of the extracted DNA or RNA.

[0034] The detection method for the rapid qualitat...

Embodiment 4

[0091] Kit specific detection:

[0092] 1) Specific verification of the qualitative detection of Lactobacillus casei:

[0093] Using the primers and methods provided in Example 3, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium bifidum, Bifidobacterium animalis, Escherichia coli, positive control (containing The plasmid of the target gene is a template), Lactobacillus casei, feed added with Lactobacillus casei; the negative control (sterilized water) is subjected to PCR amplification.

[0094]The results show: only lane 1 (the plasmid containing the target gene is the template), 2 (Lactobacillus casei), and 3 (feed that is added with Lactobacillus casei) produced a specific 415bp amplification band, while other bacterial strains did not , showing that the primers provided by the present invention can only specifically detect Lactobacillus casei. Have the amplification result of swimmin...

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Abstract

The invention discloses a rapid qualitative and quantitative detection kit, detection method and application for Lactobacillus casei added in feed. Through comparison and analysis of the reported Lactobacillus casei genome sequence, the invention provides a method for cheese milk The primers for qualitative and quantitative detection of bacilli are 5'-TGCGGCGGTAAAGGTTG-3', 5'-CGTCTGTGTAGAAACTGCGAATG-3', and the invention provides a pretreatment method for feed samples, so that the probiotics in the feed can be fully released, accurate and real reactions The number of probiotics in the feed. The invention can simply, quickly and accurately qualitatively and quantitatively detect Lactobacillus casei in pig feed without performing pure culture of probiotics, and has simple detection procedures, high detection efficiency, good accuracy, high sensitivity and good repeatability.

Description

technical field [0001] The invention belongs to the technical field of animal husbandry and veterinary medicine. More specifically, the present invention relates to a rapid qualitative and quantitative detection kit, detection method and application for Lactobacillus casei added in feed. Background technique [0002] With the prohibition of antibiotics in animal feed in my country, the development of new green additives has become a hot spot. At present, probiotic preparations are constantly developing, but the qualitative and quantitative detection methods are not scientific and fast enough, which limits the development of probiotic preparations to a certain extent. Usually, the traditional methods of physiological and biochemical reactions and selective medium pure culture isolation and counting are very easy to limit the detection efficiency and unstable detection results due to differences in bacterial species. In addition, traditional methods will also consume a lot of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12R1/245
CPCC12Q1/689
Inventor 吴涛侯永清赵迪丁斌鹰易丹王蕾陈洪波
Owner HUBEI HAOHUA BIOTECH
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