In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.
- MeSH
- DNA Probes chemistry metabolism MeSH
- Fluoresceins chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Nucleic Acids metabolism MeSH
- RNA, Viral metabolism MeSH
- Taq Polymerase metabolism MeSH
- Influenza A virus genetics MeSH
- Foot-and-Mouth Disease Virus genetics MeSH
- Phase Transition MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The foot and mouth disease virus (FMDV) causes a vesicular and contagious disease of cloven-hoofed animals. In this study, the virus was isolated from vesicles of the infected cattle using cell culture and serotyped by ELISA test. The extracted RNA from the infected cells was reverse transcribed and amplified using VP1 gene-specific primer pairs by means of one-step RT-PCR. The purified VP1 gene was sub-cloned into the uniqe KpnI and BamHI cloning sites of the pcDNA3.1+ vector. The DH5α strain of E. coli was transformed by the vector. The sequences of sub-cloned FMDV type O/IRN/2007 VP1 were aligned with FMDV type O/UKG/2001 VP1 using MegAlign software. Nucleotide sequence comparisons were made using the BLAST software available from the NCBI website. The amino acid sequences of three sub-cloned FMDV type O/IRN/2007 VP1 were also aligned with three other similar sequences using MegAlign software. Nineteen of the most similar VP1 nucleotide sequences (by BLASTN program), FMDV O/IRN/2007 VP1 sequence, twenty isolates of FMDV-O VP1 in Iran and eight topotypes of FMDV type O were aligned by Mega5 to create a FMDV-O VP1-based sequence similarity tree. The nucleotide sequence comparison indicated that FMDV O/ IRN/2007 VP1 had the greatest nucleotide sequence similarity to the VP1 gene of FMDV O1/Manisa/Turkey/69 (99%), FMDV O1/Manisa/Netherlands (98%) and FMDV O1/Manisa/iso87/Turkey (98%). It was also observed that the highest identity between FMDV O/IRN/2007 VP1 sequence and other nucleotide sequences of FMDV type O VP1 genes isolated in Iran during 1997-2004 was about 91%.
- MeSH
- Phylogeny MeSH
- Molecular Sequence Data MeSH
- Amino Acid Sequence MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA * MeSH
- Sequence Homology, Nucleic Acid MeSH
- Sequence Alignment MeSH
- Cattle MeSH
- Sus scrofa MeSH
- Genes, Viral genetics MeSH
- Capsid Proteins chemistry genetics MeSH
- Foot-and-Mouth Disease Virus genetics isolation & purification MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Iran MeSH
- MeSH
- Antigens, Viral analysis genetics immunology MeSH
- Cattle MeSH
- Foot-and-Mouth Disease epidemiology etiology prevention & control MeSH
- Viral Vaccines therapeutic use MeSH
- Foot-and-Mouth Disease Virus genetics immunology isolation & purification MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Comparative Study MeSH
- MeSH
- Enzyme-Linked Immunosorbent Assay standards MeSH
- Laboratories standards MeSH
- Antibodies, Viral enzymology MeSH
- Cattle MeSH
- Foot-and-Mouth Disease prevention & control veterinary MeSH
- Vaccination MeSH
- Foot-and-Mouth Disease Virus MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Comparative Study MeSH
