Multiple fluorescence PCR detection kit and detection method for clostridium difficile toxin genes
A Clostridium difficile toxin and multiple fluorescence technology, applied in the field of multiple fluorescent PCR detection kits for Clostridium difficile toxin gene
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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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