Reagent for detecting brucella and complex probe fluorescence quantitative PCR (polymerase chain reaction) brucella detection method
A Brucella, fluorescence quantitative technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of many influencing factors, poor method sensitivity, easy cross-contamination, etc. Specificity, ease of operation, and the effect of shortening detection time
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Embodiment 1
[0018] The preparation of embodiment 1 primer and probe
[0019] 1. Target gene selection
[0020] Brucella Cell Surfacial Protein 31kD (BCSP31) exists on the cell surface of the bacteria. This protein is encoded by the BCSP31 gene, which is species-specific and highly conserved. It can be used as a nucleic acid diagnostic marker for Brucella , according to the genbank database retrieval, the gene sequence was obtained.
[0021] 2. Design and screening of PCR primers
[0022] Following the principle of compound probe design, two sets of primers and probes were designed according to the BCSP31 gene sequence.
[0023] The 5' end of the fluorescent probe is labeled with fluorescent molecule FAM as a reporter group, and the 3' end is labeled with phosphate to block its extension. A quenching group Dabcyl is connected to the 3' end of the quenching probe.
[0024] In order to screen out a combination with high amplification efficiency from two sets of compound probe and primer ...
Embodiment 2
[0035] Embodiment 2, the detection of Brucella
[0036] In order to investigate the practical application ability of the detection of the present invention, a simulated blood specimen contaminated by Brucella was specially prepared, and pure bacteria were set as a positive control, and non-brucella pathogenic bacteria were used as a negative control.
[0037] 1. Sample Preparation
[0038] (1).Preparation and processing of contaminated blood: fixed-value Brucella was serially diluted with anticoagulant blood to make a concentration of 1×10 6 CFU / ml-1×101 CFU / ml of contaminated blood samples. Take 1ml of blood containing different concentrations of bacteria, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 1ml of red blood cell lysate (50mmol / L TrisHCL, 25mmol / L KCl, 5mmol / LMgCl 2 , pH7.5, TKM solution), shake vigorously for 2 minutes, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 1ml of TKM solution, mix well, centrifuge at 12000rpm f...
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