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Q fever Coxiella burnetii TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method

A fluorescence quantitative, fluorescent dye technology, applied in the direction of fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems affecting the reliability of detection results, etc., to achieve improved sensitivity, improved accuracy, and accurate detection. Effect

Inactive Publication Date: 2013-05-15
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, since PCR is a sensitive qualitative detection method, if the detection sample or reagent is contaminated by a small amount of target DNA, it is easy to produce false positive results, which seriously affects the reliability of the detection results.

Method used

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  • Q fever Coxiella burnetii TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method
  • Q fever Coxiella burnetii TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method
  • Q fever Coxiella burnetii TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of Q-heat Benacoxa standard plasmid

[0041] 1 Experimental materials

[0042] 1.1 Bacterial fluid, nucleic acid, vector and host

[0043] The standard strain of Q fever has been disclosed in Chen C et al., 2009 Clin.Microbiol.infect.15:2:156-157, and its strains are preserved by our laboratory. PGEM-T Easy vector was produced by Promega, and DH5α was purchased from Beijing Tiangen Biotechnology Co., Ltd.

[0044] 1.2 Reagents and kits

[0045]

[0046]

[0047] 1.3 Preparation of main reagents

[0048] (1) LB liquid medium:

[0049] Tryptone 10g

[0050] Yeast Extract 5g

[0051] NaCl 10g

[0052] Dissolve the above reagents in 900mL deionized water, adjust the pH to 7.0 with 5mol / L sodium hydroxide, adjust the volume to 1000mL, sterilize at 121°C for 20min, and store at 4°C.

[0053] (2) Ampicillin Amp stock solution (100 mg / mL): 1 g of ampicillin was dissolved in 10 mL of double-distilled water, sterilized by filtration through a ...

Embodiment 2

[0138] Example 2 Establishment of TaqMan Fluorescent Quantitative PCR Detection Method for Q-heat Benacoxella

[0139] 1 Experimental materials

[0140] 1.1 Bacterial solution, nucleic acid

[0141] The liquid with positive plasmids after sequencing was prepared from Example 1. The DNAs of Rickettsia conorii, Rickettsia rickettsii, Rickettsii mooseri and Rickettsia prowazekii are all preserved by our laboratory.

[0142] 1.2 Reagents and instruments

[0143]

[0144] 2 methods

[0145] 2.1 Design and synthesis of primers

[0146] According to the published insertion sequence IS1111 of Q-heat Benacoxa, the primers and probes for fluorescent quantitative PCR were designed using Primer premer5.0 software. Primers and probes were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0147] FF1: 5'-GGGGTAAAGTGATCTACAC-3'

[0148] FR1: 5'-CCCATAAACGTCCGATAC-3

[0149] P: 5'-FAM-TCAGTATGTATCCACCGTAGCCA-Eclipse-3'

[0150] Amplified product fragment size: 130bp

[015...

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Abstract

The invention provides a Q fever Coxiella burnetii TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method. A forward primer is 5'-GGGGTAAAGTGATCTACAC-3'; a reverse primer is 5'-CCCATAAACGTCCGATAC-3; and a probe is 5'-TCAGTATGTATCCACCGTAGCCA-3, wherein the 5' end of the probe is marked with a reporter fluorescent dye, and the 3' end is marked with a quenching fluorescent dye. The invention establishes a fluorescent quantitative PCR detection method which can conveniently, quickly and accurately detect Q fever Coxiella burnetii, is beneficial to distinguish between Coxiella burnetii and other pathogens, and is of far reaching importance in Q fever diagnosis of humans and animals.

Description

technical field [0001] The invention relates to a method for detecting Q-heat Benacox bodies, in particular to a TaqMan fluorescent quantitative PCR detection method for Q-heat Benacox bodies. Background technique [0002] Q fever (Q fever) is a zoonotic natural foci disease caused by the infection of Coxiella bumetii. It is designated as a Class B animal infectious disease, and it is listed as a Class II animal disease in my country. [0003] Benacoxiella (Coxiella bumetii) is the pathogen of Q fever, and it is also an important biological warfare agent and bioterrorist agent. It mainly enters the body in the form of aerosol through the respiratory tract to cause infection in humans and animals. Q fever generally has no specific clinical symptoms, and the diagnosis mainly depends on laboratory tests, including serological tests and the isolation and identification of pathogens. The detection of specific antibodies is generally 2 weeks after the onset, so serological testin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/11G01N21/64
Inventor 贾广乐林祥梅韩雪清王晓楠梅琳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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