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Compositions and kits and methods for detecting hypervirulent strains and/or toxin types of Clostridium difficile

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

Active Publication Date: 2018-12-14
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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  • Compositions and kits and methods for detecting hypervirulent strains and/or toxin types of Clostridium difficile
  • Compositions and kits and methods for detecting hypervirulent strains and/or toxin types of Clostridium difficile
  • Compositions and kits and methods for detecting hypervirulent strains and/or toxin types 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 3500 PCR machine and so on.

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

[0130] Targeting the TPI gene, GluD gene, tcdA gene, tcdB gene, cdtA gene, cdtB gene, and tcdC gene of C. difficile high toxic mutations (including 117 site deletion, 18bp deletion, 36bp deletion, 39bp deletion, and 54bp deletion) as targets, Design specific primers and Taqman (including MGBNFQ modified) probes.

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

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

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

[0134] tcdC△117...

Embodiment 2

[0185] Example 2: Specific detection

[0186] 1. Strains

[0187] See the table below for strains and numbers:

[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 dysentery

CMCC51105

Shigella dysenteriae

CMCC51105

Enterobacter cloacae

CMCC45301

Toxigenic Escherichia coli

CMCC44814

Salmonella typhimurium

CMCC50013

Bacteria vulnificus

CICC10383

Staphylococcus aureus

ATCC25923

Hemorrhagic Escherichia coli

ATCC12900

Enterobacter sakazakii

ATCC29544

[0189] Among them, Clostridium difficile 1-3 (ATCC BAA1804, ATCC43598, ATCC9689) are positive for tcdA and tcdB; Clostridium difficile 4 (ATCC BAA1805) is positive for tcdA, tcdB, cdtA, cdtB and contain...

Embodiment 3

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

[0221] 1. Specimen source

[0222] Among 48 feces collected from Hubei Provincial Tumor Hospital, the fecal DNA of 8 specimens that confirmed the isolation of Clostridium difficile using the CCFA selective culture method was used in this example. The specific process of the CCFA selective culture method is as follows: the anal swab specimens are mixed with an equal volume of 98% ethanol for 30 minutes to 1 hour, and 0.1 mL of the mixed solution is inoculated on the cycloserine cefoxitin mannitol agar medium, and the medium is placed at 35℃ After anaerobic incubation for 24 hours, the frozen stool is thawed and suspended in a 9-fold capacity anaerobic transport medium. For the colonies detected on the culture medium, C. difficile was determined from the traits and Gram stain.

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

[0224] Weigh 200 mg of the stool s...

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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 genes, 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. It is recognized worldwide as the most important pathogenic microorganism for hospital infections and antibiotic-induced diarrhea. It can cause antibiotic-related diarrhea, colitis, and fatal pseudomembranous enteritis. In 2013, Clostridium difficile was listed as the top three pathogens of antibiotic-related drug-resistant bacteria by the US Department of Health, and the threat level was designated as the "most urgent". my country has included the study of Clostridium difficile in the "Twelfth Five-Year Plan" national major scientific and technological ...

Claims

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Application Information

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