Composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile

A technology of Clostridium difficile and highly virulent Clostridium difficile, which is applied in the field of detection of highly virulent strains and/or toxin types of Clostridium difficile, and can solve the problems of tcdC protein truncation and inability to produce functions

Active Publication Date: 2015-09-23
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, other C. difficile high-grade strains contain 36bp deletion, 54bp deletion, etc., which all lead to truncation of tcdC protein and cannot produce function

Method used

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  • Composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile
  • Composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile
  • Composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1. Sensitivity detection

[0127] 1. Materials and Instruments

[0128] pUC57 plasmid (purchased from GenScript Biotechnology Co., Ltd.), dNTPs (purchased from TaKaRa Company), Taq enzyme (purchased from TaKaRa Company), 10×PCR buffer (purchased from TaKaRa Company), Bio-Rad PCR instrument, ABI 3500PCR instrument, etc.

[0129] 2. Design and synthesis of primers and probes

[0130] TPI gene, GluD gene, tcdA gene, tcdB gene, cdtA gene, cdtB gene, tcdC gene hypertoxic mutations (including 117-site deletion, 18bp deletion, 36bp deletion, 39bp deletion, 54bp deletion) of Clostridium difficile as targets, Design specific primers and Taqman (including MGBNFQ modification) probes.

[0131] Among them, the detection primers and probes for tcdC gene 117 site deletion are:

[0132] tcdC△117 upstream primer: 5'-TTGCTCTACTGGCATTTATTTGG-3', SEQ ID NO.1,

[0133] tcdC△117 downstream primer: 5'-ACCATGGTTCAGCATCAGACAA-3', SEQ ID NO.2,

[0134] tcdCΔ117 probe: 5'-FAM / GAAGC...

Embodiment 2

[0185] Embodiment 2: Specific detection

[0186] 1. Strains

[0187] The strains and numbers are shown in the table below:

[0188] strain

Numbering

strain

Numbering

Clostridium difficile 1

ATCC BAA1804

Clostridium difficile 2

ATCC43598

Clostridium difficile 3

ATCC9689

Clostridium difficile 4

ATCC BAA1805

Escherichia coli

ATCC8739

Bacillus subtilis

ATCC6633

Shigella flexneri

CMCC44149

Pathogenic Escherichia coli

CMCC44149

Enterobacter aerogenes

ATCC13048

Shigella dysenteriae

CMCC51105

Shigella dysenteriae

CMCC51105

Enterobacter cloacae

CMCC45301

Toxigenic Escherichia coli

CMCC44814

Salmonella typhimurium

CMCC50013

Bacteria vulnificus

CICC10383

Staphylococcus aureus

ATCC25923

Hemorrhagic E. coli

ATCC12900

Enterobacter sakazakii

ATCC29544

[0189] Among them, Clostridium difficile...

Embodiment 3

[0220] Example 3: Detection of clinical stool samples by real-time fluorescent PCR method

[0221] 1. Specimen source

[0222] Among the 48 cases of feces collected from Hubei Cancer Hospital, the fecal DNA of 8 specimens whose Clostridium difficile was confirmed to be isolated by the CCFA selective culture method was used in this example. The specific process of the CCFA selective culture method is as follows: anal swab specimens were mixed with 98% ethanol in equal volumes for 30 minutes to 1 hour, and 0.1 mL of the mixed solution was inoculated on cycloserine cefoxitin mannitol agar medium, and the medium was placed at 35°C After 24 hours of anaerobic culture, the frozen stool was thawed, and then suspended in 9 times the volume of anaerobic transport medium. For the colonies detected on the medium, C. difficile was identified from traits and Gram stainability.

[0223] 2. Use Tiangen Feces Genomic DNA Extraction Kit to extract bacterial genomic DNA

[0224] Weigh 200 mg...

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PUM

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Abstract

The invention discloses a composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile. The composition and kit comprise primers, probes and standard substances, wherein the nucleotide sequence of the primers is as shown in SEQ ID NO. 1-47; the high-virulence bacterial strains and/or toxin type of clostridium difficile can be detected through fluorogenic quantitative PCR by adopting the composition or kit. According to the invention, the sensitivity and specificity are high; the toxin type and virulence of clostridium difficile can be accurately judged through detecting tcdC gene mutation or deletion and coordinating with virulence gene detection, including tcd A/B and binary virulence gene cdt A/B, of clostridium difficile.

Description

technical field [0001] The invention belongs to the field of rapid molecular diagnosis of antibiotic-resistant Clostridium difficile gene, and specifically relates to a composition, a kit and a method for detecting high-virulence strains and / or toxin types of Clostridium difficile. Background technique [0002] Clostridium difficile (Clostridium difficile, CD) is a Gram-positive strict anaerobic bacillus with a spore structure, and is recognized worldwide as the most important pathogenic microorganism in nosocomial infections and antibiotic-induced diarrhea. It can cause antibiotic-associated diarrhea, colitis, fatal pseudomembranous colitis, etc. In 2013, Clostridium difficile was listed by the US Department of Health as the first of the three major pathogens of antibiotic-related drug-resistant bacteria, and the threat level was designated as "most urgent". my country has incorporated the research on Clostridium difficile into the National Major Science and Technology Pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/689C12Q2600/16
Inventor 殷雷张良禄曾蕾郭骁
Owner WUHAN AIMISEN LIFE TECH CO LTD
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