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

A Lactobacillus acidophilus, quantitative detection technology, applied in the field of animal husbandry and veterinary medicine, can solve the problems of inconsistent 16sRNA gene copy number, unable to truly reflect the number of microbial colonies, etc., and achieves low detection standards, good accuracy, and simple detection procedures. Effect

Active Publication Date: 2019-02-22
HUBEI HAOHUA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another problem in the qualitative and quantitative detection of probiotics in feed is the selection of the target gene. At present, most of the qualitative and quantitative detection of microorganisms select the highly conserved 16sRNA gene as the target gene, but the same gene that has been reported Genome sequence analysis of a variety of microorganisms found that the copy number of the 16sRNA gene contained in the same microorganism is inconsistent, such as Escherichia coli as an example, the currently reported copy number of the 16sRNA gene in the genome of Escherichia coli ranges from 4 to 8, Therefore, when the 16sRNA gene is used as the target gene for quantitative detection, the copy number of the 16s RNA gene cannot truly reflect the number of microbial colonies. In view of this, the present invention systematically analyzes and compares the genome sequences of probiotics that have been reported so far, and selects A single-copy gene with a conserved sequence is used as the target gene to detect probiotics, so that the copy number of the target gene can truly reflect the number of microbial colonies

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Selection of target detection genes for Lactobacillus acidophilus:

[0029] In the present invention, all genome sequences of Lactobacillus acidophilus and other probiotics that may be added to feed such as Lactobacillus casei, Lactobacillus plantarum, Lactobacillus bulgaricus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium, etc. A large number of comparative analysis, select the target gene for qualitative and quantitative detection, the target gene is a conserved housekeeping gene in the Lactobacillus acidophilus genome, and it is a single-copy gene, and specific primers are designed based on the conserved sequence of the gene , to conduct qualitative and quantitative detection of Lactobacillus acidophilus in the feed, the data obtained can truly reflect the quantity of Lactobacillus acidophilus added in the feed. The primers designed for the target gene are: 5'-CGTCCAAGTTATTTCCCCTTCA-3' and 5'-CCAGCACCTAATACTTCAAATCCAAC-3'; these primers can be used for...

Embodiment 2

[0031] A rapid qualitative and quantitative detection kit for Lactobacillus acidophilus added in feed, the kit includes primers 5'-CGTCCAAGTTATTTCCCCTTCA-3' and 5'-CCAGCACCTAATACTTCAAATCCAAC-3'.

[0032] Example: 3:

[0033] A detection method for the rapid qualitative and quantitative detection kit of Lactobacillus acidophilus added in feed, including a method for pretreatment of feed samples, the method includes:

[0034]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.

[0035] The remaining steps utilize the primers in Example 1 to carry out qualitative and quantitative identification of the extracted DNA or RNA.

[0036] The detection met...

Embodiment 4

[0097] Kit specific detection:

[0098] 1) Specificity verification of the qualitative detection of Lactobacillus acidophilus:

[0099] Utilize the primer and the method that embodiment 3 provides, to: Lactobacillus casei; Lactobacillus plantarum; Lactobacillus bulgaricus; Enterococcus faecium; Enterococcus faecalis; The plasmid of the target gene is a template); Lactobacillus acidophilus; feed added with Lactobacillus acidophilus; negative control (sterilized water) for PCR amplification.

[0100] The results show that only lane 9 (the plasmid containing the target gene is a template), 10 (Lactobacillus acidophilus), and 11 (feed added with Lactobacillus acidophilus) produced a specific 256bp amplification band, while other strains None, indicating that the primers provided by the present invention can only specifically detect Lactobacillus acidophilus. At the same time, the amplification result of swimming lane 11 shows that the sample pretreatment method provided by the p...

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Abstract

The invention discloses a rapid qualitative and quantitative detection kit, a detection method and an application for Lactobacillus acidophilus added in feed, and by comparing and analyzing the genome sequences of Lactobacillus acidophilus reported, the invention provides Primers 5'-CGTCCAAGTTATTTCCCCTTCA-3', 5'-CCAGCACCTAATACTTCAAATCCAAC-3' for the qualitative and quantitative detection of Lactobacillus acidophilus. At the same time, the invention provides a pretreatment method for feed samples, so that the probiotics in the feed can be fully released and accurately It truly reflects the number of probiotics in the feed. The invention can simply, quickly and accurately qualitatively and quantitatively detect Lactobacillus acidophilus 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 acidophilus 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/06
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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