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A method and application of Taqman probe fluorescent quantitative PCR to detect Bartonella

A real-time fluorescence quantification and probe technology, which is applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Efficient amplification and easy result determination

Active Publication Date: 2022-04-01
浙江安维珞诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned method for detecting pathogens is effective to a certain extent, but the shortcomings of PCR detection such as false positive and false negative limit its clinical application

Method used

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  • A method and application of Taqman probe fluorescent quantitative PCR to detect Bartonella
  • A method and application of Taqman probe fluorescent quantitative PCR to detect Bartonella
  • A method and application of Taqman probe fluorescent quantitative PCR to detect Bartonella

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Experimental program
Comparison scheme
Effect test

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 to find a specific diagnostic target for Bartonella. Using Primer Express 3.0 design software, fluorescent quantitative primers and Probes, synthesized by the company. The Roche LightCycler96 real-time fluorescent quantitative PCR instrument was used to monitor the real-time situation in the quantitative PCR process, and to analyze the melting curves, 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

[0032...

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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Abstract

The invention provides a primer pair and a probe for real-time fluorescent quantitative PCR detection of Bartonella, the sequence of the primer pair is shown in SEQ ID NO.1-SEQ ID NO.2; the sequence of the probe is shown in SEQ ID As shown in NO.3, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a fluorescence quenching group. The invention also provides a real-time fluorescent quantitative PCR detection kit and detection method for Bartonella, the kit including the primer pair and the probe. The present invention designs a set of specific PCR primers and TaqMan probes for specific gene regions, and establishes a Bartonella real-time fluorescence quantitative PCR identification and detection kit with good specificity and high sensitivity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a real-time fluorescent quantitative PCR detection kit and detection method for Bartonella. Background technique [0002] Bartonella is a class of facultative intracellular parasitic Gram-negative small bacilli or small cocci. In the phylogenetic classification, the genus Bartonella includes the genus Bartonella in the order Rickettsia in the Bergey Handbook of Systematic Bacteriology (Bartonella) and Rochalimaea (Rochalimaea). Before 1990, it was only known that B. baoilliformis (B. baoilliformis) and infection by B. quintana (B. quintana) could cause human disease, but more than 10 new species of B. quintana have been found so far. Physical energy makes people sick. Human infection with Bartonella can lead to trench fever, cat claw disease, myocarditis, optic retinitis and other diseases. Bacillus-like Bartonella with a single flagella, Rosalima with pili. DALY...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2561/101C12Q2563/107
Inventor 张祺梅益刘为勇刘芳刘映乐祝成亮
Owner 浙江安维珞诊断技术有限公司
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