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 sondy chemie metabolismus MeSH
- fluoresceiny chemie MeSH
- fluorescenční barviva chemie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- nukleové kyseliny metabolismus MeSH
- RNA virová metabolismus MeSH
- Taq-polymerasa metabolismus MeSH
- virus chřipky A genetika MeSH
- virus slintavky a kulhavky genetika MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- fylogeneze MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA * MeSH
- sekvenční homologie nukleových kyselin MeSH
- sekvenční seřazení MeSH
- skot MeSH
- Sus scrofa MeSH
- virové geny genetika MeSH
- virové plášťové proteiny chemie genetika MeSH
- virus slintavky a kulhavky genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Írán MeSH
- MeSH
- antigeny virové analýza genetika imunologie MeSH
- skot MeSH
- slintavka a kulhavka epidemiologie etiologie prevence a kontrola MeSH
- virové vakcíny terapeutické užití MeSH
- virus slintavky a kulhavky genetika imunologie izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- ELISA normy MeSH
- laboratoře normy MeSH
- protilátky virové enzymologie MeSH
- skot MeSH
- slintavka a kulhavka prevence a kontrola veterinární MeSH
- vakcinace MeSH
- virus slintavky a kulhavky MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
