Fluorescence labeling multiplex PCR detection kit for Clostridium difficile

A detection kit and technology for Clostridium difficile, which are applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of low sensitivity of quantitative PCR, no multiple PCR amplification, etc., to prevent misjudgment. Effect

Active Publication Date: 2014-07-09
江苏安科华捷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The scheme provided by Chinese patent application 201310108285.6 can achieve a sensitivity of 1 copy when detecting a single gene, but it does not give the technical inspiration of how to perform multiplex PCR amplification
It can be seen that the quantitative PCR sensitivity of multiple amplification is lower than that of single detection

Method used

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  • Fluorescence labeling multiplex PCR detection kit for Clostridium difficile
  • Fluorescence labeling multiplex PCR detection kit for Clostridium difficile
  • Fluorescence labeling multiplex PCR detection kit for Clostridium difficile

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design of the kit

[0042] Download the primer design template sequence from NCBI genebank, as shown in Table 4.

[0043] Table 4 Template sequence list

[0044] Locus

Sequence Genebank Number

Location

tpi

AM180355.1

3706953-3707696

tcdA

JQ809335.1

1-8133

tcdB

AF217292.1

1-7512

ctdC

HQ596359.1

1-1006

cdtA

AF271719.1

1-1392

cdtB

AF271719.1

1445-4075

[0045] The sequence of each locus is aligned in genebank, and the most conserved region is selected to design primers.

[0046] Taking the primer design of TcdA as an example, the full sequence of the primer design template JQ809335.1 was aligned in NCBI blast, and the sequence specificity of nt4000-6000 was determined to be high. Using this partial sequence as the primer design template, 4 primer sets were designed, as follows table.

[0047] Table 5 Initially designed primers for TcdA

[0048] The primers are synthesized by Shanghai Shenggong, and a single pair of primers are amplified by anneali...

Embodiment 3

[0095] Thirteen stool samples were donated by the Zhejiang Center for Disease Control and Prevention. The strains were identified and analyzed at the Zhejiang Center for Disease Control and Prevention by strain culture method and single-plex quantitative PCR.

[0096] The fecal genome was extracted using Qiagen’s QIAamp DNA stool Mini Kit, according to the operating instructions, and the template was quantified at a concentration of 0.5-2ng / μl.

[0097] The 13 fecal genomic DNAs were amplified according to the primer set, amplification system, and amplification program finally determined in Example 1, and tested as in Example 1. The software analyzes that the peak height is higher than 200 rfu, and it is considered that the corresponding gene has been amplified. On the contrary, if the peak height of the analysis result is less than 200rfu, it is considered that there is no amplification, that is, it does not contain the corresponding gene.

[0098] The 13 samples were classified in...

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Abstract

The invention provides a fluorescence labeling multiplex PCR detection kit for identification of a Clostridium difficile strain and analysis of toxin genes. The kit comprises 6 pairs of fluorescently-labeled primers used for detecting the Clostridium difficile strain. The 6 pairs of fluorescently-labeled primers comprise specific primers of specific triosephosphate-isomerase (tpi) gene, toxin A gene (tcd A), toxin B gene (tcd B), tcd C gene (tcd C), binary toxin A gene (cdt A) and binary toxin B gene (cdt B) of the Clostridium difficile strain. Amplification results are detected through capillary electrophoresis. Thus, existence of Clostridium difficile can be rapidly identified and toxin typing can be rapidly determined.

Description

Technical field [0001] The invention relates to a kit for detecting Clostridium difficile (Clostridium difficile) strains or toxic Clostridium difficile strains, and a method for detecting and identifying the presence or absence of Clostridium difficile in a sample and typing of toxins. Background technique [0002] Clostridium difficile is a gram-positive spore-producing anaerobe. Clostridium difficile itself is not invasive. Some toxin-producing bacteria cause antibiotic-related diarrhea, colitis and even fatal pseudomembranous enteritis by secreting toxin A, toxin B and binary toxins, collectively referred to as Clostridium difficile infection (Clostridium difficile infection, CDI). Among the pathogens identified in hospital-acquired infectious diarrhea, Clostridium difficile is the most common; among the causes of antibiotic-related diarrhea, Clostridium difficile also accounts for 20%-30%; and pseudomembranous enteritis is almost 100% caused by Clostridium difficile Caused...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2537/143C12Q2563/107
Inventor 葛斌文葛海鹏卢青周丽萍郑卫国
Owner 江苏安科华捷生物科技有限公司
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