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Fast qualitative and quantitative detection kit for enterococcus faecalis added to feed, detection method and application

A technology for quantitative detection of Enterococcus faecalis, applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem that qualitative and quantitative detection methods are not scientific enough and fast, and limit the development of probiotic preparations , consume a lot of time and resources, etc., to achieve the effect of low detection standard, high sensitivity and good accuracy

Active Publication Date: 2016-02-10
武汉泛华生物技术有限公司
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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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  • Fast qualitative and quantitative detection kit for enterococcus faecalis added to feed, detection method and application
  • Fast qualitative and quantitative detection kit for enterococcus faecalis added to feed, detection method and application
  • Fast qualitative and quantitative detection kit for enterococcus faecalis added to feed, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Selection of target detection genes for Enterococcus faecalis:

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

Embodiment 2

[0029] Rapid qualitative and quantitative detection kit for Enterococcus faecalis added in feed, including primers: 5'-CGTCCAAGTTATTTCCCCTTCA-3', 5'-CCAGCACCTAATACTTCAAATCCAAC-3'.

[0030] Example: 3:

[0031] The detection method of the rapid qualitative and quantitative detection kit for Enterococcus faecalis added in feed, including the method of feed sample pretreatment, 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. The detection method for the rapid qualitativ...

Embodiment 4

[0094] Kit specific detection:

[0095] 1) Specificity verification of qualitative detection of Enterococcus faecalis:

[0096]Utilize the primer and the method that embodiment 3 provides, to Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus casei, Enterococcus faecium, Bifidobacterium bifidum, Bifidobacterium animalis, Escherichia coli, positive control (containing The plasmid of the target gene is a template), Enterococcus faecalis, the feed added with Enterococcus faecalis; the negative control (sterilized water) is subjected to PCR amplification.

[0097] The results show that only lane 4 (the plasmid containing the target gene is the template), 5 (Enterococcus faecalis), and 6 (feed added with Enterococcus faecalis) produced a specific 473bp amplification band, while other strains did not , indicating that the primers provided by the present invention can only specifically detect Enterococcus faecalis. At the same time, the ampl...

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Abstract

The invention discloses a fast qualitative and quantitative detection kit for enterococcus faecalis added to a feed, a detection method and an application. Through comparative analysis of reported dung enterococcus genome sequence, the invention provides primers 5'-CGTCCAAGTTATTTCCCCTTCA-3' and 5'-CCAGCACCTAATACTTCAATCCAAC-3' for quantitative detection of the enterococcus faecalis, and meanwhile, the invention provides a pretreatment method of a feed sample, so that probiotics in the feed can be fully released; and the number of the probiotics in the feed is accurately and really reflected. The fast qualitative and quantitative detection kit can simply, quickly and accurately carry out qualitative and quantitative detection on the enterococcus faecalis in a pig feed on the basis of no probiotics cultivation, and is simple in detection program, high in detection efficiency, good in accuracy, high in sensitivity and good in 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 Enterococcus faecalis 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/689
Inventor 吴涛侯永清赵迪丁斌鹰易丹王蕾陈洪波
Owner 武汉泛华生物技术有限公司
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