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A fluorescent-labeled multiple PCR Clostridium difficile detection kit

A detection kit and fluorescent labeling technology are applied in the field of fluorescent labeling multiplex PCR Clostridium difficile detection kits, which can solve the problems of low sensitivity of quantitative PCR and no multiple PCR amplification, and achieve the effect of preventing misjudgment

Active Publication Date: 2016-02-24
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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  • A fluorescent-labeled multiple PCR Clostridium difficile detection kit
  • A fluorescent-labeled multiple PCR Clostridium difficile detection kit
  • A fluorescent-labeled multiple PCR Clostridium difficile detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The design of embodiment 1 kit

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

[0043] Table 4 Template sequence list

[0044] loci

Serial Genebank number

Location

tpi

AM180355.1

3706953-3707696

tcA

JQ809335.1

1-8133

tcdB

AF217292.1

1-7512

ctdC

HQ596359.1

1-1006

cdT

AF271719.1

1-1392

cdB

AF271719.1

1445-4075

[0045] The sequences of each locus were compared in genebank, and the most conserved regions were selected to design primers.

[0046] Taking the primer design of TcdA as an example, the complete sequence of the primer design template JQ809335.1 was compared on NCBIblast, and it was determined that the sequence specificity of nt4000-6000 was high, and 4 pairs of primer sets were designed using this part of the sequence as the primer design template, as shown in the following table .

[0047] Table 5 P...

Embodiment 3

[0095] The 13 feces samples were donated by the Zhejiang Center for Disease Control and Prevention. The bacterial strains were identified and analyzed by the strain culture method and single-weight quantitative PCR detection in the Zhejiang Provincial Center for Disease Control and Prevention.

[0096] The fecal genome was extracted with the QIAampDNAstoolMiniKit kit from Qiagen Company, according to the operating instructions, and the quantified concentration of the template was 0.5-2 ng / μl.

[0097] The 13 stool genomic DNAs were amplified according to the primer set, amplification system, and amplification program finally determined in Example 1, and detected by the detection method in Example 1. If the software analysis peak height is higher than 200rfu, it is considered that the corresponding gene is amplified. On the contrary, if the peak height of the analysis result is lower than 200rfu, it is considered that there is no amplification, that is, the corresponding gene i...

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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 strains or toxic Clostridium difficile strains, and a method for detecting and identifying Clostridium difficile in a sample and typing the toxin. Background technique [0002] Clostridium difficile is a Gram-positive spore-forming anaerobic bacterium. Clostridium difficile itself is not invasive, and some toxin-producing bacteria cause antibiotic-associated diarrhea, colitis, and even fatal pseudomembranous enteritis by secreting toxin A, toxin B, and binary toxins, collectively referred to as Clostridium difficile infection (CDI) . Clostridium difficile is the most common pathogen identified in hospital-acquired infectious diarrhea; Clostridium difficile also accounts for 20% to 30% of the causes of antibiotic-associated diarrhea; and pseudomembranous enteritis is almost 100% caused by Clostridium difficile due to. Clostridium difficile infection is the number one cause of nosocomi...

Claims

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Application Information

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Patent Type & Authority Patents(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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