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PCR specific primer and molecular beacon TaqMan probe combination for simultaneously detecting two anaplasma and detection method

A molecular beacon, no slurry technology, applied in the field of molecular biology, to achieve high sensitivity, reduce environmental pollution, and avoid product pollution.

Pending Publication Date: 2020-01-03
HENAN UNIV OF ANIMAL HUSBANDRY & ECONOMY
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  • Abstract
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  • Application Information

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Problems solved by technology

Duplex real-time quantitative PCR for goat aplasma and phagophilia aplasma has not been reported so far

Method used

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  • PCR specific primer and molecular beacon TaqMan probe combination for simultaneously detecting two anaplasma and detection method
  • PCR specific primer and molecular beacon TaqMan probe combination for simultaneously detecting two anaplasma and detection method
  • PCR specific primer and molecular beacon TaqMan probe combination for simultaneously detecting two anaplasma and detection method

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Embodiment Construction

[0042] The present invention will be further elaborated below in conjunction with the examples to facilitate a better understanding of the present invention, but not to limit the present invention.

[0043] 1. Primers and Molecular Beacons Taq Man probe design

[0044] Use Primer Express software to design primers for goat aplasma groEL Gene (National Center for Biotechnology Information (NCBI) database, reference sequence: KM206275) and Phagocytophilia aplasma 16S rRNA Gene (National Center for Biotechnology Information (NCBI) database, reference sequence: HQ629911) designed primers and molecular beacons Taq The Man probe (see Table 1) was then synthesized by Shanghai Sangon Biotechnology Co., Ltd. The specificity of each pair of primers was evaluated using the BLAST alignment tool (BLAST) of the National Center for Biotechnology Information (NCBI) database (https: / / www.ncbi.nlm.nih.gov / ).

[0045] Table 1 Primers and Molecular Beacons Taq Man probe information

[...

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Abstract

The invention discloses a PCR specific primer and molecular beacon TaqMan probe combination for simultaneously detecting two anaplasma and a detection method. The 5' end of a probe of anaplasma capraand anaplasma phagocytophilum is connected with a fluorescence reporting group (FAM or Cy5), the 3' end of the probe of the anaplasma capra and the anaplasma phagocytophilum is connected with a fluorescence quenching group (BHQ3 or BHQ1), the size of an amplification product of a special primer of the anaplasma capra groEL gene locus is 153 bp, and the size of an amplification product of a specialprimer of the anaplasma phagocytophilum 16S rRNA gene locus us 81 bp. A dual real-time quantitative PCR reaction system is 25 [mu]L of a PCR reaction system, and comprises 12.5 [mu]L of Premix Ex Taq, 0.4 [mu]M of each primer group, 0.4 [mu]M of probe DNA, 2 [mu]L of a template, and sterile deionized water is complemented to 25 [mu]L. The reaction condition of the dual real-time quantitative PCRamplification is a two-step method: 95 DEG C 30s, 95 DEG C 5s, 55.5 DEG C 30s, and operation is circulated for 40 times. The advantages of high detection speed, high sensitivity, high specificity, high efficiency, low cost and the like are achieved, and especially the absolute quantification of pathogens in a sample can be achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a combination of PCR special primers and a molecular beacon TaqMan probe for simultaneous detection of two kinds of aplasma, and a detection method. Anaplasma capra ) and phagophilic aplasma ( A. phagocytophilum ). [0002] technical background [0003] Anaplasmosis is a disease of the genus Anaplasma (Anaplasmataceae) Anaplasma ) is a tick-borne disease caused by aplasma infection of host blood cells. At present, 7 kinds of aplasma have been found at home and abroad, namely, phagophilic aplasma ( Anaplasma phagocytophilum ), without pulp at the edge ( A. marginale ), central aplasma ( A. centrale ), bovine anhydrous ( A. bovis ), flat anhydrous ( A. platys ), sheep anhydrous ( A. ovis ) and goat anhydrous ( A. capra ). Among them, goat aplasma and phagophilic aplasma have a very wide host spectrum, including humans, and are the only two aplasma that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/113
Inventor 彭永帅李月勤张利卫赵姗姗王军张曼
Owner HENAN UNIV OF ANIMAL HUSBANDRY & ECONOMY
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