Digital PCR method for detecting brucella nucleic acid DNA

A Brucella nucleic acid and Brucella technology, applied in the field of molecular biology, can solve the problems of high false positive rate and false negative rate, affecting application, etc., and achieve the effect of high sensitivity and sensitive applicability

Inactive Publication Date: 2020-10-02
ICDC CHINA CDC
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  • Abstract
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AI Technical Summary

Problems solved by technology

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, but the current fluorescent quantitative PCR technology, due to many factors affecting tissue fluid and blood , resulting in high false positive rate and false negative rate, affecting its application in the detection of Brucella nucleic acid DNA in tissue blood

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  • Digital PCR method for detecting brucella nucleic acid DNA
  • Digital PCR method for detecting brucella nucleic acid DNA
  • Digital PCR method for detecting brucella nucleic acid DNA

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Embodiment

[0032] Example Sensitivity Test

[0033] 1. The nucleic acid DNA of the sample to be tested is the nucleic acid DNA extracted by the kit;

[0034] 2. Forward primer: F1: 5'-ACCTTGCCCTTGCCATCAT-3', SEQ ID NO: 1;

[0035] Reverse primer: R1: 5'-AGTCCGGCTTTACGCAGTCA-3', SEQ ID NO: 2;

[0036] Probe sequence: Probe: 5'-TGCCGTTATAGGCCCAATAGGCAACG-3', SEQ ID NO: 3, wherein the 5' end is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group BHQ-1.

[0037] 3. The digital PCR reaction system includes: 2╳ddPCR Supermix 10μL (special enzyme system for digital PCR); nucleic acid DNA to be tested 1μL; forward primer F1 and reverse primer R1 each 1.6μL, probe Probe 0.8μL, supplemented with double distilled water to 20 μL. Wherein, the concentrations of the primer and probe solutions are both 10 μmol / L;

[0038] 4. The pretreatment process of the PCR reaction system is as follows:

[0039]1. (1) Preparation of microdroplets: 20 μL of PCR reaction sys...

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Abstract

The invention relates to a digital PCR method for detecting the brucella nucleic acid DNA, and relates to the technical field of molecular biology. A digital PCR technology in the invention can detectand identify pure brucella nucleic acid DNA, and also can detect and identify trace brucella nucleic acid DNA in tissue fluid, such as blood; the brucella nucleic acid DNA of a sample to be detectedcan be qualitatively identified; the copy number of the brucella nucleic acid DNA of the sample to be detected also can be quantitatively detected; the minimum 3.67 fg / [mu]L of the nucleic acid DNA can be detected; namely, the minimum detectable range can be one brucella nucleic acid DNA copy; in addition, digital PCR result analysis can be standardized; and data comparison of different experimenters can be carried out.

Description

technical field [0001] The invention relates to the technical field of molecular biology. The invention relates to a digital PCR method for detecting Brucella nucleic acid DNA. The method can quickly and quantitatively detect the copy number of the Brucella nucleic acid DNA in a sample to be tested. 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 known to be hosts of Brucella. The sources of infection related to humans are mainly sheep, cattle and pigs, followed by dogs. The blood and other tissues of sick animals, as well as their secretions, excreta, stream products and milk contain a large ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/04C12R1/01
CPCC12Q1/6851C12Q1/689
Inventor 田国忠
Owner ICDC CHINA CDC
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