Gexp rapid detection primer set, kit and application for simultaneous identification of main subtypes of avian influenza
A technology for detection kits and detection primers, which is applied in the determination/inspection of microorganisms, microorganisms, recombinant DNA technology, etc., can solve problems that have not been reported yet, and achieve the effects of rapid identification, rapid sensitivity, and high sensitivity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0024] Example 1 : Multiplex RT-PCR Primer Design for Avian Influenza Virus
[0025]Refer to relevant literature to download the sequences of 7 genes of avian influenza virus M, H5, H7, H9, N1, N2 and N9 from the GenBank database, and use DNAStar to analyze and compare the nucleotide sequences of each gene to find out the suitable design specific gene sequence. Conserved region of sex primer, utilize GeXP express profiler tool to design the specific primer (seeing table 1) for 7 pairs of genes of avian influenza virus, the designed primer adopts Primer Premier 5.0, NCBI PrimerBlast and Oligo7.0 to analyze and screen, then A non-homologous unique sequence was added to the 5' ends of all forward primers and reverse primers as a universal primer (Uni-Primer), and the 5' end of the upstream universal primer was labeled with a fluorescent dye Cy5, namely Cy5-Tag-F, which was determined by Synthesized by Shanghai Invitrogen Company and purified by HPLC.
[0026] Table 1 Primer in...
Embodiment 2
[0030] Example 2: Establishment of multiplex PCR detection system
[0031] 2.1 Preparation of template and monoclonal plasmid standard containing target gene
[0032] According to the instructions of TaKaRa Company MiniBEST Viral RNA / DNAExtraction Kit Ver.5.0 (catalogue number DV819A), extract nucleic acids of different subtypes (H1-16 and N1-9) of avian influenza virus and other poultry viruses, and obtain 50 μL of nucleic acid samples, and divide them into devices Store at -80°C. The RT reaction system was carried out according to the instructions of TaKaRa reverse transcriptase (catalogue number D2639A), and the obtained RNA samples were reverse-transcribed according to the following reaction system and reaction conditions to obtain cDNA; DEPC water was used as the control of total RNA.
[0033] Reaction system (25μL): 5×Reverse Transcriptase Buffer 5μL, 50mmol / L RandomPrimer (9mer) 1μL, dNTP Mixture (10mM / L) 2μL, 40U Ribonuclease Inhibitor 0.5μL, 5U / μL MLV Reverse Trans...
Embodiment 3
[0048] Example 3: GeXP system multiple detection system specific detection
[0049] According to the instructions of MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0, extract HA(1-16) and NA(1-9) subtype avian influenza virus nucleic acid from allantoic fluid, and simultaneously extract IBV, NDV and ILTV and other common poultry viruses Nucleic acids were added to the GeXP multiple detection system established in Example 2 to detect the specificity of the method. After the multiplex PCR was completed, the PCR products were analyzed by GeXP capillary electrophoresis, and the results showed that only specific signals appeared in each reaction, and there was no cross-reaction. HA gene subtypes except H5, H7, and H9, NA gene subtypes except N1, N2, and N9 subtypes, and NDV, IBV, ILTV, and blank control had no reaction signals, suggesting that the established method had strong specificity , no cross-reaction with other test objects.
PUM
Property | Measurement | Unit |
---|---|---|
Sensitivity | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com