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Dual fluorescence PCR detection kit, primer and probe for simultaneously detecting anaplasma phagocytophilum and Ehrlichia chaffeensis

A detection kit and phagocytic cell technology, which can be applied to microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problems of time-consuming and laborious success rate, complicated isolation of rickettsial pathogens, etc., and achieve the effect of improving detection efficiency.

Inactive Publication Date: 2015-01-21
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The isolation of Rickettsia pathogens is complicated, time-consuming and laborious, and the success rate is very low

Method used

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  • Dual fluorescence PCR detection kit, primer and probe for simultaneously detecting anaplasma phagocytophilum and Ehrlichia chaffeensis
  • Dual fluorescence PCR detection kit, primer and probe for simultaneously detecting anaplasma phagocytophilum and Ehrlichia chaffeensis
  • Dual fluorescence PCR detection kit, primer and probe for simultaneously detecting anaplasma phagocytophilum and Ehrlichia chaffeensis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The composition of embodiment 1 kit

[0056] 1. The composition of the kit

[0057] 1) DNA extraction reagent; Tiangen DNA extraction kit (TIANamp Genomic DNA Kit, cat.no DP304);

[0058] 2) DNA standard product: a DNA fragment of the major outer membrane protein (msp2) gene of Anaplasma phagocytophilum, as shown in the sequence listing SEQ ID No: 9; and a DNA fragment of the 16S rRNA gene of Ehrlichia chaffae, as shown in the sequence listing SEQ ID No: 12;

[0059] 3) Real-time quantitative PCR reaction solution, which includes: 2×Premix Ex Taq (Probe qPCR) mixture, 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for detecting Anaplasma phagocytophilum, and detecting Chaffee 0.2 μM sense primer, 0.2 μM antisense primer and 0.4 μM fluorescent probe for Ehricia body, 1× fluorescent quantitative PCR reference dye;

[0060] Wherein, the 2 × Premix Ex Taq (Probe qPCR) mixed solution was purchased from Takra, (RR390A), and the fluorescent quanti...

Embodiment 2

[0074] The specificity test of embodiment 2 kit

[0075] 1 material,

[0076] Negative mouse spleen and tick samples, common rickettsia and other bacterial DNA samples used for specificity evaluation came from our laboratory and the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences, including Rickettsia africana, Rickettsia moschii , Bartonella henselae, DNA from hemorrhagic E. coli, Staphylococcus aureus, plague.

[0077] 2 methods:

[0078] Utilize the kit usage method of the present invention to detect negative mouse spleen and tick samples and common rickettsia and bacterial DNA, simultaneously with 10 5 Concentration of plasmid DNA was used as a positive control to verify its specificity.

[0079] 3 results

[0080] The test results of negative mouse spleen and tick samples, common rickettsia and bacterial DNA samples were all negative, indicating that the method has no cross-reaction to the above pathogens, indicating that the sys...

Embodiment 3

[0081] Sensitivity detection of embodiment 3 kit

[0082] In order to verify the detection sensitivity of the kit of the present invention, with 10-fold diluted DNA standard substance (2×100~2×10 7 copy / μL) as the template, add 2 μL to each dilution of the template, and set up a negative control at the same time, according to the established method, carry out the amplification reaction on the quantitative PCR instrument, and obtain the kinetic curve and standard curve, when the template is 2 When ×10 copies / μL, the CT value is still within the detectable range (CT<40), indicating that the method established in this experiment can detect at least 2×10 copies / μL of DNA.

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Abstract

The invention discloses a dual fluorescence PCR detection kit, primer and probe for simultaneously detecting anaplasma phagocytophilum and Ehrlichia chaffeensis. The detection primer and probe are shown as SEQ ID NO:1 to SEQ ID NO:6 in a sequence table, wherein the sequences SEQ ID NO:1 and SEQ ID NO:2 respectively refer to a forward primer and a reverse primer for detecting the anaplasma phagocytophilum; the sequence SEQ ID NO:3 refers to a fluorescence probe for detecting the anaplasma phagocytophilum; the sequences SEQ ID NO:4 and SEQ ID NO:5 respectively refer to a forward primer and a reverse primer for detecting Ehrlichia chaffeensis; and the sequence SEQ ID NO:6 refers to a fluorescence probe for detecting the Ehrlichia chaffeensis. The invention also provides the kit for detecting the anaplasma phagocytophilum and Ehrlichia chaffeensis. The primer and probe provided by the invention are extremely high in specificity, two viruses can be detected at a time, and the efficiency is improved.

Description

technical field [0001] The invention relates to a primer, a probe and a detection kit for detecting Anaplasma phagocytophilum and Ehrlich chaffae body. Background technique [0002] Anaplasma phagocytophilum and Ehrlichia chaffae are both intracellular parasites. Anaplasma phagocytophilum infects neutrophils in peripheral blood, causing human granulocytic anaplasmosis (HGA), with fever accompanied by leukopenia, thrombocytopenia, and multiple organ dysfunction as the main clinical manifestations. Ehrlichia Chaffee infects human mononuclear macrophages, causing human monocytic ehrlichiosis (HME), which is very similar to human granulocytic anaplasmosis in clinical symptoms, characterized by fever, lymph node Swelling and appearance of atypical lymphocytes are the main clinical manifestations. Both HGA and HME are zoonotic diseases with natural foci, which are distributed worldwide and are mainly transmitted through tick bites. [0003] The clinical manifestations of rickett...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2545/101C12Q2537/143C12Q2563/107
Inventor 郭惠琳田洁刘艳华田茵赵静
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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