Specific primer and probe used for detecting shigella flexneri and real-time fluorescence quantification PCR kit
A real-time fluorescent quantification technology of Shigella flexneri, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of unfavorable finding of pathogens and a large amount of time
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Embodiment 116
[0029] The preparation of embodiment 1 16s gene standard product
[0030] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. The 16S gene widely exists in Shigella flexneri and is highly conserved. The present invention uses the 16s gene of Shigella flexneri as the target sequence. This example mainly uses PCR technology to amplify the 16s gene of Shigella flexneri, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-16s, and carry out corresponding PCR identification and analysis. Sequencing identification, and finally quantified as a standard for the method to be established, laying the foundation for the next method and evaluation.
[0031] 1. Preparation of template DNA
[0032] Genomic DNA of Sh...
Embodiment 2
[0093] Embodiment 2 real-time fluorescent quantitative PCR kit
[0094] 1. Design and synthesis of specific primers and probes
[0095]The present invention carries out bioinformatics comparative analysis to the complete sequence of Shigella flexneri 16s gene in NCBI database, selects the conserved fragment sequence suitable for designing primers and probes as the target (see Example 1), and further uses Primerexpress 3 software, Primer Premier 5 software and Oligo 7 software, designed multiple sets of real-time fluorescent quantitative PCR primers and probes, and after preliminary screening, a set of fluorescent quantitative PCR primers and probes for detecting Shigella flexneri were finally determined. probe.
[0096] As the core of the present invention, a group of primers and probe nucleotide sequences for Shigella flexneri real-time fluorescent PCR detection are as follows:
[0097] Upstream primer: 16s-415F 5'-TATAGTTTAGCTTGCCTTT-3'
[0098] Downstream primer: 16s-589...
Embodiment 3
[0118] Example 3 The quantitative detection method of the sample to be tested by the real-time fluorescent quantitative PCR kit
[0119] Respectively with the 16S gene serial concentration standard substance (the serial concentration of serial concentration standard substance of preparation of test sample DNA and embodiment 1 is: 1.00 * 10 9 copies / ml, 1.00×10 8 copies / ml, 1.00×10 7 copies / ml, 1.00×10 6 copies / ml, 1.00×10 5 copies / ml, 1.00×10 4 copies / ml, 1.00×10 3 copies / ml, 1.00×10 2 copies / ml) as a template, use the primers and probes in the kit to perform fluorescent quantitative PCR amplification, and set up positive and negative controls at the same time.
[0120] The PCR reaction system is as follows:
[0121]
[0122] Reaction conditions: 94°C pre-denaturation for 2 minutes, 94°C for 10 seconds, 60°C for 30s and collecting fluorescence signals, 40 cycles. Draw a standard curve, and perform rapid quantitative detection through the standard curve and the Ct va...
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