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Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria

A fluorescence quantification and kit technology, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms to achieve high accuracy, convenient operation, and effective detection.

Inactive Publication Date: 2018-09-07
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no published literature on the simultaneous detection of 8 common sheep viruses and bacteria by using the fluorescent quantitative PCR method with Taqman probes

Method used

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  • Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria
  • Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria
  • Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria

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Experimental program
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Effect test

Embodiment 1

[0080] Embodiment 1 (establish and detect 8 kinds of sheep common viruses and bacteria Taqman fluorescent quantitative PCR detection system simultaneously)

[0081] Design primers and Taqman probes:

[0082] According to the 8 kinds of pathogenic sequences registered in NCBI, the sequences were compared by DNAstar software to find out the specific conserved sequences of various pathogenic gene sequences. Primer Primer 5.0 software was used to design primers and probes. Multiple pairs of specific primers and probes were obtained, and after comparison and screening, a set of optimal primers and MGB probes were finally determined.

[0083] Table 1.8 kinds of pathogenic primer probe sequence list

[0084]

[0085] Viral RNA extraction and reverse transcription:

[0086] (1) Take out the Peste small ruminant disease virus (PPRV) that needs to be extracted in advance;

[0087] (2) Take out two 1.5mL EP tubes in the ultra-clean room and add 200μl virus;

[0088] (3) After add...

Embodiment 2

[0114] Embodiment 2 (preparation of common sheep virus and bacteria Taqman fluorescent quantitative PCR kit and sample detection)

[0115] Kit preparation:

[0116] Reagent 1 preparation: see Table 5 and Table 6;

[0117] Table 5. Fluorescence quantitative PCR reaction solution A configuration table (400μL)

[0118] Reagent

Usage amount

PPRV-F

10μL

PPRV-R

10μL

PPRV-Taqman

5μL

ORFV-F

30μL

ORFV-R

30μL

ORFV-Taqman

15μL

GTPV-F

10μL

GTPV-R

10μL

GTPV-Taqman

5μL

C.a-F

30μL

C.a-R

30μL

C.a-Taqman

15μL

Sterile deionized water

200μL

[0119] Table 6. Fluorescence quantitative PCR reaction solution B configuration table (400μL)

[0120]

[0121]

[0122] Reagent 2: 500 μL of 2× enzyme premixed reaction solution;

[0123] Reagent 3:

[0124] Positive control A (PPRV, ORFV, GTPV, C.a positive plasmid mixture) 100 μL;

[0...

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Abstract

The invention discloses a Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria, and relates to the technical field of molecular biology. The eight common sheep viruses and bacteria include peste des petits ruminants virus (PPRV), orf virus (ORFV), goatpox virus (GTPV), chlamydophila abortus (C.a), staphylococcus aureus (S.a), escherichia coli (E.coli), streptococcus (Stre.) and salmonella (Salm.). PCR reaction liquid comprises eight groups of primers and eight groups of Taqman probes. The kit can rapidly and effectively detect8 sheep pathogens simultaneously, the detection method is accurate, the specificity and the sensibility are high, the stability is good, and it can be achieved that to-be-detected pathogens is rapidly diagnosed and effectively detected.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a Taqman fluorescent quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria. Background technique [0002] my country is a big country in agriculture and animal husbandry, and my country's mutton production ranks first in the world. In recent years, sheep breeding has changed from a small-scale, free-range breeding model to a large-scale standardized house feeding model, resulting in a trend of increasing scale breeding year after year, and the change in breeding methods has also led to the infectiousness of sheep. A large number of diseases occur, and the problem is becoming more and more obvious, and it is often caused by simultaneous infection of multiple pathogens, which makes it very difficult to diagnose and prevent the occurrence of diseases. [0003] Common infectious disease pathogens in sheep include Peste des pet...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12Q1/689C12Q1/14C12Q1/10C12Q1/04C12N15/11C12R1/93C12R1/445C12R1/19C12R1/46C12R1/42C12R1/01
CPCC12Q1/6851C12Q1/689C12Q1/701C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 许信刚张琪付明哲杨峰
Owner NORTHWEST A & F UNIV
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