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Nested PCR detection method or/and identification method of brucella

A Brucella, to-be-detected technology, applied in the field of molecular biology, can solve the problems of complex operation process, high false positive rate and false negative rate, difficult detection, etc., and achieve high sensitivity, good specificity, and simple test operation. Effect

Inactive Publication Date: 2019-02-05
ICDC CHINA CDC
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  • Claims
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AI Technical Summary

Problems solved by technology

The trace amount of nucleic acid DNA in the sample to be tested is difficult to detect by ordinary PCR and multiplex PCR methods with a single primer. For pure bacterial DNA, it is difficult to identify the biotype of Brucella with a single primer. The process is complicated, and trace amounts of DNA cannot be detected; fluorescent quantitative PCR technology is a nucleic acid quantitative detection technology that integrates PCR technology, fluorescent signal detection and data analysis
It has high specificity, sensitivity, accuracy and low false positive rate for pure bacterial DNA, and can detect 1 copy number of nucleic acid DNA. , resulting in high false positive rate and false negative rate, affecting its application in the detection of Brucella nucleic acid DNA in tissue blood

Method used

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  • Nested PCR detection method or/and identification method of brucella
  • Nested PCR detection method or/and identification method of brucella
  • Nested PCR detection method or/and identification method of brucella

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Embodiment 1

[0048] In the embodiment of the present invention, the nested PCR simultaneously detects and identifies Brucella species and biotypes. First, according to the transposase orfA and transposase orfB gene sequences registered in GenBank, the Primer 5.0 software is used to design the nested PCR first and second respectively. Secondary amplification primers.

[0049] Primers for the first PCR amplification:

[0050] Forward primer: F1: 5'-AACGTAACCAGATCATAGCGCATG-3', as shown in SEQ ID NO: 1;

[0051] Reverse primer: R1: 5'-ACAGATGAGCAATGGAACCGGAT-3', as shown in SEQ ID NO: 2;

[0052] Primers for the second PCR amplification:

[0053] Forward primer: F2: 5'-GAATGCGGTCAATGTTTTCTCGC-3', as shown in SEQ ID NO: 3;

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Abstract

The invention relates to a nested PCR detection method or / and an identification method of brucella, and relates to the technical field of molecular biology. The nested PCR technique of the invention can not only detect and identify pure bacterial nucleic acid DNA, but also detect and identify trace brucella nucleic acid DNA of tissue fluid, blood, and the like; and can not only identify brucella species of the nucleic acid DNA of the sample to be tested, but also identify the biotype thereof. The nested PCR result analysis can be standardized, with simple test operation, and can be completed by a person with ordinary PCR techniques.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, and the present invention relates to detection and identification Brucella bacterial strain and the nested PCR technique method of Brucella DNA in the detection sample (as: blood, serum, interstitial fluid, liver spleen etc.), by Sequence the PCR amplification products to identify the Brucella species and biotypes. The method can be used to quickly diagnose the brucellosis infection from the etiology, and identify the brucella species and biotypes. Background technique [0002] Brucellosis, referred to as brucellosis, also known as geothermal relaxation fever, Malta fever or wave fever, commonly known as "lazy man's disease", is a disease caused by intracellular parasitic bacteria of the genus Brucella. A zoonotic infection—allergic disease, brucellosis is widely distributed all over the world. [0003] More than 60 kinds of livestock, poultry, and wild animals are currently k...

Claims

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Application Information

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IPC IPC(8): C12Q1/6848C12Q1/04G16B40/00C12R1/01
CPCC12Q1/6848C12Q2531/113C12Q2549/119
Inventor 田国忠姜海朴东日赵鸿雁杨晓雯
Owner ICDC CHINA CDC
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