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A kind of Clostridium gordii specific PCR detection primer and method

A clostridium gordonii and detection primer technology, applied in the field of clostridium gordonii-specific PCR detection primers, can solve the problems of less strain research, less molecular level research, limited PCR technology, etc., and achieve easy determination and detection results. special effect

Active Publication Date: 2020-03-17
SHIHUIDA PHARMA GRP (JILIN) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the genome sequence of Clostridium gordii has not yet been detected, and there are few studies on this strain, the relevant research is limited to the overall application of the strain, and there are few studies on its molecular level, which limits the application of PCR technology to detect it. to develop

Method used

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  • A kind of Clostridium gordii specific PCR detection primer and method
  • A kind of Clostridium gordii specific PCR detection primer and method
  • A kind of Clostridium gordii specific PCR detection primer and method

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Embodiment 1

[0039] The development of embodiment 1 clostridium gordii specific PCR detection primers

[0040] The inventor found through the previous sequencing of the whole genome of Clostridium gordii and the analysis of the sequencing results that the bacterium contains a gene sequence encoding thioredoxin, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0041] Using DNAMAN software comparison, it was found that the thioredoxin gene sequence of this bacterium had little similarity with Clostridium Novyi, Clostridium beijerinckii, Clostridium sporogenes and other Clostridium anaerobic bacteria thioredoxin gene sequence similarity, respectively. 25.23%, 34.60% and 33.38%, which are different from the highly conserved rule of Clostridium anaerobic thioredoxin gene sequence. According to the high specificity of the gene sequence encoding Clostridium gordii thioredoxin, the primers for specific amplification of Clostridium gordii are designed and synthesized.

Embodiment 2

[0043] Step 1, designing and synthesizing Clostridium gordii-specific PCR detection primers, the nucleotide sequence is as follows:

[0044]F: 5'-ATGGCTAAAACAATAAATACAGGGA-3'; SEQ ID NO.1

[0045] R: 5'-TTATAAATGAGCTTCTACTTTTTGCT-3'; SEQ ID NO.2

[0046] Step 2, RNA template preparation

[0047] Clostridium gordii was inoculated in 6ml RCM liquid medium (purchased from BD Company), and cultured in an anaerobic workstation at 37°C for 12 hours, then 1mL of the bacterial liquid was taken, centrifuged at 6000r / min for 5min, the supernatant was discarded, and 100μL Sterilized double-distilled water, gently blowing with a pipette, resuspended the bacteria; put the bacteria liquid into a mortar fully pre-cooled with liquid nitrogen, grind to powder, and add 1mL Trizol for RNA extraction.

[0048] Step 3, reverse transcription of RNA into cDNA

[0049] According to TaKaRa PrimeScript TM The RT-PCR kit (RR014A) kit instruction manual was used to carry out reverse transcription to ...

Embodiment 3

[0057] Embodiment 3PCR detection method specificity evaluation test

[0058] According to the method described in Example 2, cDNA templates were prepared and PCR was detected, and PCR amplification reactions were performed on bacteria in samples such as Clostridium gordoi, Escherichia coli, stomach, and intestine.

[0059] Specific test results such as figure 1 as shown, figure 1 The results showed that only Clostridium gordii had a specific band at 315bp, while no specific band appeared in other lanes.

[0060] Sensitivity evaluation test of PCR detection method

[0061] Inoculate Clostridium gordii in 6mL RCM liquid medium, culture in an anaerobic workstation at 37°C for 12 hours, take 1ml of the bacterial solution, and perform 10-fold serial dilution with sterile 1×PBS buffer solution, and count the bacteria by plate method. Concentration is 10 8 CFU / mL. Take 1mL of bacterial liquid to extract RNA according to the example, perform reverse transcription, and detect the ...

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Abstract

The invention relates to clostridium ghonii specific PCR detection primers and a method. Nucleotide sequence of a forward primer of the pair of clostridium ghonii specific PCR detection primers is shown as SEQ ID NO.1, and nucleotide sequence of a reverse primer is shown as SEQ ID NO.2. According to the invention, it is discovered for the first time that an encoding gene Trx of thioredoxin obeys to a highly conserved law which is different from other thioredoxin gene sequences, which keep a close genetic relationship, such as clostridium novyi, clostridium beijerinckii, clostridium sporogene and the like; and on the basis of the finding, the clostridium ghonii specific PCR detection primers are designed, so that technical difficulties in clostridium ghonii in-vivo distribution and clinical detection by virtue of a PCR technology are solved.

Description

technical field [0001] The invention relates to a specific PCR detection primer and method for Clostridium gordii, belonging to the technical field of biological detection. Background technique [0002] Clostridium ghonii is a Gram-positive, strictly anaerobic bacterium that can exist in soil, water sediment, animal and human intestines. At present, the research of Clostridium gordii is mainly focused on the use of its hypoxic property in the tumor (it can only multiply in a large number in the hypoxic area of ​​the tumor), and secretes a variety of hydrolytic enzymes, induces the body's anti-tumor immune response, and inhibits the growth of tumor tissue. , to carry out the research and development of new drugs such as bacterial oncolytic drugs. [0003] Chinese patent document CN104473973A (application number 201410803072.X) discloses the application of a domesticated Clostridium gordoi MW-DCG-HNCv-18 strain in the preparation of drugs for the treatment of non-small cell l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12Q1/689C12Q1/04C12N15/11C12R1/145
CPCC07K14/33C12Q1/689
Inventor 王勇刘园园李莲莲王丹果双双杨阳朱红徐兴鲁邢立超邓广霞韩停邵石丽黄琦位杨冬霞张西坤
Owner SHIHUIDA PHARMA GRP (JILIN) LTD
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