Clostridium ghonii specific PCR detection primers and method

A Clostridium gordii and detection primer technology, which is applied in the field of Clostridium gordii-specific PCR detection primers, can solve the problems of less research on bacterial strains, less research at the molecular level, and limited PCR technology, and achieve specific and easy detection results. Judgmental effect

Active Publication Date: 2017-06-20
SHANDONG XINCHUANG BIOLOGICAL TECH CO LTD
View PDF7 Cites 0 Cited by
  • 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 t

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Clostridium ghonii specific PCR detection primers and method
  • Clostridium ghonii specific PCR detection primers and method
  • Clostridium ghonii specific PCR detection primers and method

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0039] Example 1 Development of specific PCR detection primers for Clostridium gordonii

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

[0041] Using DNAMAN software comparison, it was found that the thioredoxin gene sequence of this bacterium is not closely related to Clostridium Novyi, Clostridium beijerinckii, Clostridium sporogenes, and other clostridium anaerobes. The similarity is only 25.23%, 34.60% and 33.38%, which are different from the rule that the thioredoxin gene sequence of anaerobic Clostridium is highly conserved. According to the high specificity of the gene sequence encoding Clostridium gordonii thioredoxin, primers for specific amplification of Clostridium gordonii were designed and synthesized.

Example Embodiment

[0042] Example 2

[0043] Step 1: Design and synthesize PCR detection primers specific to Clostridium gordonii. The nucleotide sequence is as follows:

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

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

[0046] Step two, RNA template preparation

[0047] Inoculate Clostridium gordonii in 6ml RCM liquid medium (purchased from BD company), culture it in an anaerobic workstation at 37°C for 12h, take 1ml of bacterial solution, centrifuge at 6000r / min for 5min, discard the supernatant, and add 100μL Sterilize double-distilled water, pipette gently to resuspend the bacteria; place the bacteria liquid in a mortar fully pre-cooled with liquid nitrogen, grind to powder, and add 1 mL of Trizol for RNA extraction.

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

[0049] Follow TaKaRa PrimeScript TM RT-PCR kit (RR014A) kit instructions for reverse transcription to prepare cDNA template, and determine the cDNA template concen...

Example Embodiment

[0057] Example 3 Evaluation Test of Specificity of PCR Detection Method

[0058] Prepare cDNA template and PCR detection according to the method described in Example 2, and perform PCR amplification reaction on bacteria in samples such as Clostridium gordonii, E. coli, stomach and intestine.

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

[0060] PCR detection method sensitivity evaluation test

[0061] Inoculate Clostridium gordonii in 6mL RCM liquid medium, place it in an anaerobic workstation at 37°C for 12 hours, take 1ml of bacterial solution, dilute it with sterile 1×PBS buffer 10 times, and count the bacteria by plate method. The concentration is 10 8 CFU / mL. Take 1 mL of bacterial solution to extract RNA according to the example, perform reverse transcription, and detect the cDNA concentration. Dilute the cDNA with sterile doub...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/31C12Q1/68C12Q1/04C12N15/11C12R1/145
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 SHANDONG XINCHUANG BIOLOGICAL TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap