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

A quantitative detection and kit technology, 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 and rapid, limit the development of probiotic preparations, It takes a lot of time and resources to achieve the effect of low detection standard, high sensitivity and good accuracy

Active Publication Date: 2018-05-29
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 bulgaricus added in feed
  • Rapid qualitative and quantitative detection kit, detection method and application of Lactobacillus bulgaricus added in feed
  • Rapid qualitative and quantitative detection kit, detection method and application of Lactobacillus bulgaricus added in feed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Selection of Lactobacillus bulgaricus target detection genes:

[0027] The present invention has all the advantages of Lactobacillus bulgaricus that have been reported and other probiotics such as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus bulgaricus, Enterococcus faecalis, Enterococcus faecalis, Bifidobacterium and other probiotics that may be added to the feed. A large number of comparison and analysis were carried out on the genome sequence of the bacterium, and the target gene for qualitative and quantitative detection was selected. The target gene is a conserved housekeeping gene in the Lactobacillus bulgaricus genome, and it is a single-copy gene. Based on the conserved sequence of the gene, a specific The qualitative and quantitative detection of Lactobacillus bulgaricus in the feed is carried out by using the primers of nature, and the data obtained can truly reflect the quantity of Lactobacillus bulgaricus added in the...

Embodiment 2

[0029] A rapid qualitative and quantitative detection kit for Lactobacillus bulgaricus added to feed, including primers: 5'-TCGAAATGCTGAATCTGCCTAA-3', 5'-TCGTCATCGTCTCTTCTGTAAAACC-3'.

[0030] Example: 3:

[0031] A detection method for a rapid qualitative and quantitative detection kit for Lactobacillus bulgaricus added to feed, including a method for pretreatment of feed samples, the method includes:

[0032] Mix 25g of fully pulverized and mixed 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. Perform aseptic operation on the uniform dilution prepared in the previous step by 10-fold incremental dilution, select more than 2 to 3 suitable dilutions, and extract bacterial genomic DNA or RNA as needed.

[0033] The remaining steps utilize the primer pairs in Example 1 to perform qualitative and quantitative identification of the extracted DNA or RNA.

[0034] The detection method of the rapid ...

Embodiment 4

[0091] Kit-specific detection:

[0092] 1) Specificity verification of Lactobacillus bulgaricus qualitative detection:

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

[0094] The results showed that only lane 8 (the plasmid containing the target gene as a template), 9 (Lactobacillus bulgaricus), and 10 (feed supplemented with Lactobacillus bulgaricus) produced a specific 450bp amplified band, while other strains did not , indicating that the primers provided by the present invention can only specifically detect Lactobacillus bulgaricus. Simultaneously there is the amplificatio...

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Abstract

The invention discloses a rapid qualitative and quantitative detection kit, a detection method and an application for Lactobacillus bulgaricus added in feedstuffs. By comparing and analyzing the genome sequences of Lactobacillus bulgaricus reported, the invention provides The primers for qualitative and quantitative detection of bacilli are 5'-TCGAAATGCTGAATCTGCCTAA-3', 5'-TCGTCATCGTCTCTTCTGTAAACC-3', and the invention provides a feed sample pretreatment method, so that the probiotics in the feed can be fully released, accurate and true reaction The number of probiotics in the feed. The invention can simply, quickly and accurately qualitatively and quantitatively detect Lactobacillus bulgaricus 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 bulgaricus added to feed. Background technique [0002] With the banning 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 their 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 method of separation and counting of pure culture of physiological and biochemical reaction and selective medium is used, which is very easy to limit the detection efficiency and unstable detection results due to differences in bacterial species. In addition, traditional methods also...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/06C12R1/225
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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