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

Inactive Publication Date: 2011-08-17
浙江国际旅行卫生保健中心
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Problems solved by technology

But so far there is not a mature method for the detection of tularemia, the main reason is that tularemia is highly pathogenic and difficult to isolate and cultivate
[0003] The discovery of traditional PCR technology and nucleic acid hybridization technology has greatly promoted the development of bacterial detection technology, but there are defects such as easy cross-contamination and incapable of quantitative analysis.

Method used

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  • Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis
  • Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis
  • Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis

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Effect test

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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Abstract

The invention provides a reagent for detecting francisella tularensis. The reagent comprises an upstream primer, a downstream primer, a fluorescent probe and a quenching probe, wherein the gene sequence FTF1 of the upstream primer is 5'-aagcaagtgttgactacagat-3', the gene sequence FTR1 of the downstream primer is 5'-caccaaagaaccatgttaaac-3', the gene sequence FPFT1 of the fluorescent probe is 5'-FAM-aatcatgttagtacccgctctgcca-p-3', and the gene sequence QPFT1 of the quenching probe is 5'-agcgggtactaacatgat-Dabcyl-3'. The invention also provides a complex probe and fluorescent quantitative PCR method for detecting the francisella tularensis by using the reagent. Compared with the traditional francisella tularensis detecting technology, the invention has the advantages of simple operation, high efficiency, quickness and high specificity.

Description

technical field [0001] The invention relates to a reagent for detecting Francisella tularensis and a detection method for Francisella tularensis, in particular to a method for amplifying a marker gene of Francisella tularensis with a composite probe, and then detecting Francisella tularensis by using a fluorescent PCR technique of the composite probe. Background technique [0002] Francisella tularensis (Francisella tularensis) is a Gram-negative coccus that can cause tularemia in humans and animals. It is one of the most infectious pathogenic bacteria and has been found in nature. More than a hundred species of animals are infected with this bacterium. Because of its diverse transmission routes, easy diffusion, and strong toxicity, it is listed as a Class A bioterrorist agent by the US Centers for Disease Control and Prevention. The fatality rate of tularemia is very high. In the absence of effective treatment, the most virulent type A tularemia subspecies can be fatal if ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 吕沁风郑伟吴忠华杨永耀李禾
Owner 浙江国际旅行卫生保健中心
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