Primers and method for multiplex fluorescent quantitative PCR (polymerase chain reaction) detection of avian metapneumovirus typing
A multiple fluorescence quantitative, avian metapneumovirus technology, applied in the field of molecular biology detection of viruses, can solve the problem that other subtypes cannot be detected, and achieve good accuracy and detection efficiency, good repeatability, and strong specificity. Effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0036] Design and synthesis of primers and probes
[0037] According to the coding gene SH sequence of aMPV A and B subtype SH proteins published in GenBank and the P gene nucleotide sequence of aMPV-C, the Snapgene software was used to design specific primers and probes. The primer sequences are shown in Table 1 below (see gene sequence list):
[0038] Table 1 The sequences of the above primers and probes were all specific to aMPVA and subtype B by BLAST analysis
[0039]
[0040] sexual sequence.
Embodiment 2
[0042] Establishment of qPCR detection method (1) Preparation of standard positive template
[0043] The aMPV-A SH gene was synthesized by the applicant and connected to the vector. The cDNA of the IT / Ty / A / 259-01 / 03 strain (GenBank accession number JF424833.1) was used as a control for the aMPV-A type, and the aMPV-B type With the cDNA of the VCO3 / 60616 strain (GenBank accession number AB548428.1) as a control, the aMPV-C type uses the cDNA of the Colorado strain (GenBank accession number AY590688) as a control, and uses the primers designed in Example 1 to carry out PCR amplification, The reaction system is 20 μL: including 10 μL of 2×KOD One PCR Master Mix, 1.0 μL of upstream and downstream primers, 1.0 μL of template, ddH 2 Make up 20 μL with O; the amplification program is: 98°C for 30s, 98°C for 15s, 55°C for 5s, 68°C for 5s, 30 cycles; 68°C for 5min.
[0044] The PCR amplification products were identified by 1.5% agarose gel electrophoresis, and the electrophoresis resu...
experiment example 1
[0067] Clinical Sample Testing
[0068] Use the real-time fluorescence quantitative PCR method and conventional PCR method that the present invention establishes to carry out fluorescence quantitative PCR reaction detection Ct value respectively to 105 chicken nasopharyngeal swabs of suspected aMPV infection sick chicken with optimized reaction system and condition, and compare with traditional common PCR test results were compared.
[0069] As can be seen from the above results, the present invention has established a multiplex fluorescent quantitative PCR method based on the SH genes of aMPV-A and aMPV-B and the P gene of aMPV-C. This method only specifically amplifies aMPV-A, B and C subtypes, does not cross-react with NDV, hMPV, IBV, ILTV, NTC, etc., and has high specificity. The minimum detection number of this method is 1 copy, which is 10 copies lower than that of the fluorescent quantitative PCR method for subtypes A, B, and C established by Yun Bingling. 3 Copies / μL...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More