Primer for simultaneously detecting pasteurellamultocida and capsular A type dual real-time fluorescentquantitative PCR method of pasteurellamultocida and application

A real-time fluorescence quantitative, Pasteurella technology, applied in the biological field, can solve the problems of false positives, difficult to buy, strict requirements for target bacteria, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2017-09-15
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the bacterial isolation and identification method is very complicated in actual operation, and is easily affected by the interference of various bacteria. The requirements for the content of bacteria in the bacterial liquid and the number of miscellaneous bacteria are relatively strict, and there may be false negative and false positive results
The typing of bacterial capsules mainly adopts serological methods and capsule PCR typing: serological methods require capsule type standard serum, which is difficult to buy and takes time and effort

Method used

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  • Primer for simultaneously detecting pasteurellamultocida and capsular A type dual real-time fluorescentquantitative PCR method of pasteurellamultocida and application
  • Primer for simultaneously detecting pasteurellamultocida and capsular A type dual real-time fluorescentquantitative PCR method of pasteurellamultocida and application
  • Primer for simultaneously detecting pasteurellamultocida and capsular A type dual real-time fluorescentquantitative PCR method of pasteurellamultocida and application

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Experimental program
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Effect test

specific Embodiment approach 1

[0018] Specific embodiment one: a kind of primers and application of double real-time fluorescent quantitative PCR method for simultaneous detection of Pasteurella multocida and its capsular type A of the present embodiment, described primers include upstream and downstream primers and Taqman probe primers ,details as follows:

[0019] Upstream primer Kmt-F: 5'-GCTYGTTGTGAGTGGGCTTGT-3';

[0020] Downstream primer Kmt-R: 5'-GCCGTTGTCAAGGAAGCAGAT-3';

[0021] Probe primer Kmt-P: 5'FAM-TTTGTTGGGCRGAGTTTGGTG-BHQ1 3';

[0022] Upstream primer CapA-F: 5'-GGGCTGGCTATGTTGGTTTAT-3';

[0023] Downstream primer CapA-R: 5'-CTTTATCTGTGATGGGCGATT-3';

[0024] Probe primer CapA-P: 5'HEX-TAGCTCAACACCACAATGTGATCT3'.

specific Embodiment approach 2

[0025] The PCR reaction system is 20 μL;

[0026]

[0027]

[0028] PCR reaction conditions: 95° C. for 5 min; 95° C. for 15 sec, 53° C. for 20 sec, 72° C. for 30 sec, 35 cycles; fluorescent signal detection was performed during the extension of the cycle.

[0029] One difference between this embodiment and the specific embodiment is that the primers are used to detect Pasteurella multocida and its capsule type A. Others are the same as in the first embodiment.

[0030] The content of the present invention is not limited to the content of the above-mentioned embodiments, and a combination of one or several specific embodiments can also achieve the purpose of the invention.

[0031] Verify the beneficial effects of the present invention through the following examples:

Embodiment 1

[0033] Design of dual real-time fluorescent quantitative PCR primers for Pasteurella multocida and its capsular type A

[0034] Referring to the kmt1 sequence of the Pasteurella multocida Pm70 strain recorded in GenBank (GenBank No.: AE004339), the sequence alignment of the reference strains was carried out with the help of bioinformatics software, and the gene sequences of the kmt1 and hyaD-hyaC genes of Pasteurella multocida were analyzed and obtained. Conserved area. ABI PrimerExpress 3.0 real-time fluorescent quantitative PCR primer design software was used to design and synthesize TaqMan probes and primers. Two pairs of primers and probes were designed.

[0035] Simultaneously detect the primers and the detection method of the double Real-time PCR method of Pasteurella multocida and its capsule A type, the primers include upper and lower primers and probe primers, as follows:

[0036] Upstream primer Kmt-F: 5'GCTYGTTGTGAGTGGGCTTGT 3', as shown in SEQ ID NO.1;

[0037] ...

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Abstract

The invention discloses a primer for simultaneously detecting pasteurellamultocida and a capsularA type dual real-time fluorescentquantitative PCR method of the pasteurellamultocida and application, and relates to the technical field of biology. The primer adopts Kmt-F: 5'-GCTYGTTGTGAGTGGGCTTGT-3'; Kmt-R: 5'-GCCGTTGTCAAGGAAGCAGAT-3'; Kmt-P: 5'-FAM-TTTGTTGGGCRGAGTTTGGTG- BHQ13'; CapA-F: 5'-GGGCTGGCTATGTTGGTTTAT-3'; CapA-R: 5'-CTTTATCTGTGATGGGCGATT-3';CapA-P: 5' HEX-TAGCTCAACACCACAATGTGATCT-3'. The primer simultaneously detects kmt1 and hyaD-hyaD genes.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer and application of a dual Real-time PCR method for simultaneously detecting Pasteurella multocida and its capsule type A. Background technique [0002] Pasteurella multocida (Pm) can cause a variety of diseases in humans and animals, including fowl cholera, bovine hemorrhagic septicemia, swine atrophic rhinitis, rabbit hemorrhagic septicemia, and human subcutaneous infection. Higher morbidity and mortality. Pasteurella multocida is divided into A, B, D, E, and F types according to the capsule type. The conventional method for detecting Pasteurella multocida antigen is to isolate and identify the bacteria, or to detect by ordinary PCR after enrichment. Among them, the bacterial isolation and identification method is very complicated in actual operation, and is easily affected by the interference of various bacteria. The requirements for the content of bacteria in the bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/686C12Q1/689C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143C12Q2561/101
Inventor 郭东春曲连东刘家森刘大飞姜骞
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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