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

A fluorescence quantitative and fluorescent technology, which is applied in microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve problems affecting the reliability of test results, etc., and achieve improved sensitivity, less environmental pollution, and improved accuracy. sexual 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 SYBR GreenI fluorescent quantitative PCR (polymerase chain reaction) detection method
  • Q fever Coxiella burnetii SYBR GreenI fluorescent quantitative PCR (polymerase chain reaction) detection method
  • Q fever Coxiella burnetii SYBR GreenI fluorescent quantitative PCR (polymerase chain reaction) detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of Q-heat Benacoxa standard plasmid

[0045] 1 Experimental materials

[0046] 1.1 Bacterial fluid, nucleic acid, vector and host

[0047] 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.

[0048] 1.2 Reagents and kits

[0049]

[0050]

[0051] 1.3 Preparation of main reagents

[0052] (1) LB liquid medium:

[0053] Tryptone 10g

[0054] Yeast Extract 5g

[0055] NaCl 10g

[0056] 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.

[0057] (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

[0142] Example 2 Establishment of the SYBR Green I Fluorescence Quantitative PCR Detection Method for Q-heat Benacoxia

[0143] 1 Experimental materials

[0144] 1.1 Bacterial solution, nucleic acid

[0145] 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.

[0146] 1.2 Reagents and instruments

[0147]

[0148] 2 methods

[0149] 2.1 Design and synthesis of primers

[0150] 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.

[0151] FF2: 5'-TGCTTCAGTATGTATCCAC-3'

[0152] FR2: 5'-GTTACGATCATTCTTGTTAC-3'

[0153] Amplified product fragment size: 172bp

[0154] 2.2 Preliminary establishment of SYBR Gr...

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Abstract

The invention provides primers and a method for Q fever Coxiella burnetii SYBR GreenI fluorescent quantitative PCR (polymerase chain reaction) detection. A forward primer is 5'-TGCTTCAGTATGTATCCAC-3'; and a reverse primer is 5'-GTTACGATCATTCTTGTTAC-3'. 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 method for detecting Q-heat Benacox bodies by SYBR Green I fluorescent quantitative PCR. 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 serologic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/01
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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