Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis
A fluorescence quantification, Tula technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of easy cross-contamination, difficult separation and cultivation, strong pathogenicity of Tula bacteria, etc. The effect of ensuring specificity and shortening detection time
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Embodiment 1
[0018] Example 1 Preparation of primers and probes
[0019] 1 Target gene selection
[0020] The FopA gene encodes the outer membrane protein of Francisella tularensis, and there are still anti-fopA antibodies in the serum of people in the recovery period. This gene is highly conserved in the Francisella genus and can be used as a nucleic acid diagnostic marker for Francisella tularensis. The gene sequence was obtained by searching the genbank database.
[0021] 2PCR primer design and screening
[0022] Following the principles of compound probe design, two sets of primers and probes were designed according to the FopA 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 a combination with high amplification efficiency from two sets of com...
Embodiment 2
[0036] Embodiment 2, the detection of Francisella tularensis
[0037]In order to investigate the practical application ability of the detection of the present invention, a simulated blood sample contaminated by Francisella tularensis was specially prepared, and pure bacteria were set as a positive control, and non-Francis tularensis pathogenic bacteria were used as a negative control.
[0038] 1. Sample Preparation
[0039] (1).Preparation and treatment of contaminated blood: fixed-value Francisella tularensis was serially diluted with anticoagulant blood to make a concentration of 1×10 6 CFU / ml-1×10 1 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 / LTrisHCL, 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...
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