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A kind of efficient identification/screening method and application of Clostridium butyricum

A clostridia and butyric acid technology, applied in the field of microorganisms, can solve the problems of difficulty in screening, low proportion, and difficult to obtain, and achieve the effects of improving screening efficiency, speeding up experimental process, and long screening period

Active Publication Date: 2020-12-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the specificity of the RCM agar medium is poor, and Clostridium butyricum is not the dominant strain in the intestinal tract and soil, and its relative abundance is low, which makes the final screening more difficult
The specificity of TSN agar medium is higher than that of RCM agar medium, but due to the uneven distribution of Clostridium butyricum in samples, especially in feces samples, the proportion of samples containing Clostridium butyricum is low, the workload is large and it is not easy Obtaining the target strain makes the screening and isolation of Clostridium butyricum accidental and random, with a large workload and it is not easy to obtain the target strain, which greatly reduces the chance of mining this specific strain resource

Method used

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  • A kind of efficient identification/screening method and application of Clostridium butyricum
  • A kind of efficient identification/screening method and application of Clostridium butyricum
  • A kind of efficient identification/screening method and application of Clostridium butyricum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 identifies the method for Clostridium butyricum

[0043] Design the following primers:

[0044] (1) The sequences of primers CBU21F / CBU1023R are:

[0045] Forward primer: 5'-TCAATTAGAAGGCAGAGTACC-3' (SEQ ID NO: 1)

[0046] Reverse primer: 5'-CTAAAACTGACTGTGGCATT-3' (SEQ ID NO: 2)

[0047] The size of the amplified target gene fragment is 1022bp;

[0048] (2) The sequences of primers CBU80F / CBU551R are:

[0049] Forward primer: 5'-CAAAGTCATCATCTAGTCGT-3' (SEQ ID NO: 3)

[0050] Reverse primer: 5'-TCCATTATAAGCTGGTGCAT-3' (SEQ ID NO: 4)

[0051] The size of the amplified target gene fragment is 491bp;

[0052] (3) The sequences of primers CBU64F / CBU488R are:

[0053] Forward primer: 5'-TACACTCCTATCATCACCCTTTAT-3' (SEQ ID NO:5)

[0054] Reverse primer: 5'-CACCTAAATCGGCAGCAGCAT-3'SEQ ID NO:6

[0055] The size of the amplified target gene fragment is 445bp.

[0056] Template DNA extraction: Genomic DNA was extracted from 20 Clostridium butyricum and 12 ...

Embodiment 2

[0062] Embodiment 2 screens the method for Clostridium butyricum

[0063] Based on the TSN selective medium, the screening of Clostridium butyricum in the fecal samples with the target band amplified was completed. Dilute the samples in a gradient manner, spread 100 μL of each dilution on the TSN selective medium plate, culture anaerobically at 37°C for 48 hours, select a single colony that grows normally under anaerobic conditions, and streak on the TSN selective medium plate After isolation and incubation at 37°C for 48 hours, a single colony similar in colony morphology to Clostridium butyricum was selected. Carry out PCR amplification according to the method of Example 1, and obtain the corresponding specific target band as the target strain.

Embodiment 3

[0064] Example 3 High-efficiency identification / screening of the accuracy detection of Clostridium butyricum PCR amplification primers

[0065] CBU21F / CBU1023R, CBU80F / CBU551R and CBU64F / CBU488R were used as primers to detect the accuracy of PCR amplification primers.

[0066] Extract the genomic DNA of 20 strains of Clostridium butyricum from different samples, use the optimized primers CBU21F / CBU1023R and the reaction system and reaction program in Example 1 to carry out PCR amplification, and detect the specificity of the PCR amplification primers of the present invention. sex. The results of agarose gel electrophoresis figure 1 shown, from figure 1 It can be seen that the specific primer CBU21F / CBU1023R used to amplify Clostridium butyricum in this embodiment can accurately amplify the target sequence fragments of 20 strains of Clostridium butyricum, which is a positive result, and the water control reaction No amplification band was detected, which was a negative resul...

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Abstract

The invention discloses a method for efficiently identifying / screening clostridium butyricum and application thereof, and belongs to the technical field of microorganisms. Specific primers are designed according to the core gene of the clostridium butyricum, and the specific primers are used for screening and identifying the clostridium butyricum. Compared with traditional screening methods basedon a TSN selective medium, the PCR method for efficiently screening clostridium butyricum provided by the invention is high in accuracy up tp 100%, high in specificity and high in sensitivity, and thedetection limit is 7.95x10<-3> ng / [mu]L. The method can reduce the range of screened samples, improve the probability of screening out target strains, effectively shorten the screening period, reducethe workload and improve the screening efficiency.

Description

technical field [0001] The invention relates to a method for efficiently identifying / screening Clostridium butyricum and its application, belonging to the technical field of microorganisms. Background technique [0002] Clostridium butyricum, also known as Clostridium butyricum, is a Gram-positive anaerobic bacterium belonging to the Bacillus family and the genus Clostridium. Widely present in cheese, natural yogurt, human and animal manure and soil. Clostridium butyricum produces gas during the fermentation process, and the fermentation products include acetic acid, butyric acid, butanol, amylase, lipase, vitamins, etc. The study of Clostridium butyricum as a microecological agent was first discovered and reported by a Ph.D. from Chiba Medical College in Japan in 1933. Since then, Clostridium butyricum has been widely used as a enteritis treatment drug, food additive, veterinary drug and feed additive because it can regulate the microecological balance of the human intest...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12R1/145
CPCC12Q1/689
Inventor 陈卫陆文伟易至翟齐啸崔树茂赵建新张灏
Owner JIANGNAN UNIV
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