Method for detecting Bartonella through TaqMan probe fluorescent quantitative PCR and application
A detection method, real-time fluorescent quantitative technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve problems such as false positives, false negatives, and restrictions on popularization and application, and achieve good specificity , efficient amplification, and high sensitivity
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Embodiment 1
[0029] The real-time fluorescent quantitative PCR of embodiment 1 Bartonella detects the design and screening of primer pair and probe
[0030] 1. Using the Blastn local comparison software, the nucleic acid sequence of Bartonella was analyzed and compared with nucleic acids of other pathogens, and a specific diagnostic target for Bartonella was found. Using Primer Express 3.0 design software, fluorescent quantitative primers and Probes, synthesized by the company. The Roche LightCycler96 real-time fluorescence quantitative PCR instrument was used to monitor the real-time situation in the quantitative PCR process, and to analyze the melting curve, take-off time, amplification efficiency, and the time required to reach the plateau of different primers and probe test groups. A set of fluorescent quantitative PCR primer pairs and probes with the highest amplification efficiency and the best specificity were screened out, and the sequences are shown in Table 1.
[0031] Table 1 ...
Embodiment 2
[0033] Real-time fluorescent PCR kit and detection method of embodiment 2 Bartonella
[0034] 1. The composition of the kit
[0035] This kit includes a pair of specific primers for Bartonella (as shown in Table 1), a specific fluorescent probe (as shown in Table 1), positive control, negative control, 5×PCR Buffer and Enzyme Mix;
[0036] (1) A pair of specific primers for Bartonella (upstream primers and downstream primers are shown in Table 1)
[0037] (2) A specific fluorescent probe of Bartonella (as shown in Table 1)
[0038] In this embodiment, the 5' end of the fluorescent probe is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the fluorescent quencher group BQ1; in other embodiments, other fluorescent reporter groups and other fluorescent quencher groups can also be selected. Specifically, the fluorescent reporter group can also be replaced by VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the fluorescent quencher group can als...
Embodiment 3
[0072] The specificity test of embodiment 3 Bartonella fluorescent quantitative PCR amplification
[0073] The samples used in the specificity test of the Bartonella real-time fluorescent quantitative PCR detection method have other positive strains or positive samples. After fluorescent quantitative PCR amplification, the results showed that only the Bartonella positive specimens and positive controls had amplification curves, and the other 16 pathogens and negative controls had no amplification curves ( image 3 ), see Table 1 for details. This result shows that the detection method has good specificity.
[0074] Table 1 specificity analysis of the present invention
[0075]
[0076]
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