Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes

A Clostridium difficile toxin and detection kit technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism determination/inspection, etc.

Active Publication Date: 2013-10-23
杭州海基生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has a sensitivity of 60% to 70% and a specificity of 98%

Method used

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  • Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes
  • Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes
  • Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the acquisition of standard product

[0061] 1. Materials:

[0062] The pGEM-T-Easy cloning system, PCR-related reagents and Taq DNA polymerase were purchased from Promega, USA, 377 sequencer (ABI Company), Bio-Radi cycler PCR instrument (Bio-Rad Company), ABI7500fast quantitative PCR instrument (ABI Company) company).

[0063] 2. Primer and probe design and synthesis:

[0064] Using Clostridium difficile tcdA (GenBank registration number JQ809336.1), tcdB (GenBank registration number JQ809336.1), cdtA (GenBank registration number HQ639678.1) and cdtB (GenBank registration number HQ639678.1) genes as templates , using Primer Express TM (V3.0, American ABI Company) software to analyze TaqMan primers and probe sites, and select the best combination. The primers and probes were synthesized and purified by Shanghai Huirui Biotechnology Co., Ltd.

[0065] 3. Preparation of positive reference product:

[0066] The oligonucleotide sequences were synthesized ...

Embodiment 2

[0081] Example 2: Establishment of a method for detecting four related toxin genes of Clostridium difficile by multiplex fluorescent quantitative PCR

[0082] 1. Plasmid DNA and other bacterial DNA extraction:

[0083] Use genomic DNA extraction reagent to extract bacterial genomic DNA, use plasmid DNA extraction kit to extract positive plasmid DNA, take 1.0 μL (50ng / μL) as template, and use upstream and downstream primers for detection to perform on ABI7500fast quantitative PCR instrument (ABI company) PCR amplification.

[0084] The composition of the PCR reaction solution is as follows:

[0085]

[0086] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, 40 cycles of amplification at 95°C for 15 seconds, and 45 seconds at 60°C, and finally placed at 4°C. Fluorescence acquisition was performed at the annealing temperature of each cycle.

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Abstract

A provided multiple fluorescence PCR detection kit for clostridium difficile toxin genes mainly comprises specific primers, probes and a PCR reaction reagent, and the specific primers and the probes respectively consist of specific primers and probes of clostridium difficile toxin A (tcdA), toxin B (tcdB), binary toxin A (cdtA) and binary toxinB (cdtB). The beneficial effects of the invention comprise: the fast, sensitive and specific multiple fluorescence PCR detection kit and a detection method are provided for the clostridium difficile toxin genes, and a foundation is provided for distinguishing between toxigenic strains and avirulent strains of clostridium difficile, and early diagnosis on infection of clostridium difficile.

Description

(1) Technical field [0001] The invention relates to a Clostridium difficile toxin gene multiplex fluorescent PCR detection kit and a detection method. (2) Background technology [0002] Clostridium difficile (Clostridium difficile) is a Gram-positive anaerobic bacillus, which is one of the normal flora in the human intestinal tract and is widely distributed in the natural environment and animal feces. Clostridium difficile itself is not invasive, but when taking a large amount of broad-spectrum antibiotics, immunosuppressants, proton pump inhibitors or chemotherapy drugs, some other normal intestinal flora are inhibited, and Clostridium difficile reproduces in large numbers in the intestinal tract, among which Some toxin-producing strains can cause antibiotic-associated diarrhea, colitis and even pseudomembranous colitis by secreting toxin A and toxin B. The binary toxin produced by some high-yielding virulence strains will lead to an increase in the incidence and recurrenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/145
Inventor 金大智罗芸王贤军张严峻叶菊莲
Owner 杭州海基生物技术有限公司
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