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is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method, the test has some difficulties due to the need for specially equipped laboratory and time-consuming nature in large-scale population screenings [ 1 , 4 , 5
In this study, bovine viral diarrhoea virus (BVDV) was detected and biotypically characterised in clinical samples using reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR technique produced two different amplicons (402 and approx. 680 bp in size) in case of the presence of both biotypes (cp and ncp) in the sample. The mixture of the biotypes as detected by RT-PCR was verified by the immunoplaque assay (IPA). Purification of biotypes was carried out by native plaque isolation and subsequent RT-PCR revealed single products (402 or approx. 680 bp in size) in each clone. The results showed that RT-PCR can be used for accurate molecular differentiation between the BVDV biotypes.
Classical swine fever is a highly contagious, notifiable disease of pigs and wild boars listed by the World Organisation for Animal Health (OIE). Therefore, methods employed in the diagnosis of CSF should be fast, sensitive and specific. The aim of this study was optimisation of the reverse transcription reaction to increase the sensitivity of real-time RT-PCR for the detection of classical swine fever virus, the aetiological agent of the disease. The efficiency of reverse transcription reaction was compared including a range of reverse transcriptases, thermal conditions and priming methods based on results obtained in the following realtime PCR. Depending on catalysis and the priming method used in the study a significant diversity of results was observed. The best efficacy of reverse transcription was obtained using SuperScript II reverse transcriptase and priming with random nonamers and reverse, gene-specific primer. This combination improved the sensitivity of RT-PCR nearly 1000 times as compared to the method with AMV reverse transcriptase coupled with random hexamers. In summary, this study has demonstrated that the optimisation of reverse transcription can contribute to a higher sensitivity of RT-PCR diagnostic methods.
Wall, S. J., Edwards, D. R. (2002) Quantitative reverse transcription-polymerase chain reaction (RT-PCR): A comparison of primer-dropping, competitive, and real-time RT-PCRs. Anal. Biochem. 15 , 269-273. Quantitative reverse
Differential Display RT-PCR was developed before the genomic era to serve as a tool in hunting for genes. Nowadays, applications using state-of-the-art techniques to obtain more information about the whole transcriptome or the genome have rapidly overtaken DD-RT-PCR. This paper will discuss a few of the major drawbacks and limitations of using this once highly valued method.
is very important to regularly update the commercial PRRSV RT PCR kits. Furthermore, these kits do not have discriminatory capacity, they cannot differentiate the herd-specific wild virus from the applied MLV strain ( Toplak et al., 2012 ). Therefore
A real-time RT-PCR assay utilising light upon extension fluorogenic primer (LUX RT-PCR) was developed for the rapid and efficient detection of avian influenza viruses (AIV). The assay detected each of the AIV isolates tested (16/16) and gave negative results with heterologous pathogens (17/17). The detection limit of the assay proved to be 10-0.5 EID50/0.2 ml and 101.5 EID50/0.2 ml in allantoic fluid of virus-infected embryonated chicken eggs and in spiked chicken faeces samples, respectively. Based on its specificity, sensitivity and relative simplicity, the LUX RT-PCR assay provides a novel, rapid and cost-effective diagnostic tool for avian influenza surveillance and monitoring programs.
One-step RT-PCR followed by a RFLP assay was developed for the typing of Barley yellow dwarf virus (BYDV) member of Luteovirus genera. RFLP analysis performed on 23 samples including one lab isolate showed three types of Hpa II restriction profile. A partial coat protein (CP) gene sequencing was carried out and confirmed the RFLP analysis. Both sequencing and RFLP analysis identified the presence of 3 BYDV-PAV (including the lab isolate Blatno85), 10 BYDV-MAV and 10 BYDV-PAS isolates among the field samples. One-step RT-PCR together with RFLP presents an easy and reliable assay for routine BYDV typing. These methods revealed that PAS and MAV are more dominant than PAV in the Czech Republic.
Using bioinformatic data, we found that maize homologues of the Arabidopsis single subunit RING type ubiquitin ligase genes, COP1 (Constitutive Photomorphogenesis 1) and SINAT5 (Seven in Absentia in Arabidopsis thaliana 5) have been frequently predicted during large-scale cDNA sequencing project and released to databases since 2008. Despite this general information, no tissue specific expression profiles of these genes were published to our knowledge so far. In the present research, the expression and the relative levels of COP1 and SINAT5 transcripts were detected using reverse transcription and polymerase chain reaction (RT-PCR) with template materials collected separately from three different organs (root, leaf and seed) of maize plants grown under the normal growth conditions. The results not only confirmed the presence of COP1 transcript in all three test samples but also revealed its abundance in root tissues that may be consistent to its large number of targets for ubiquitination in darkness. Analysis of SINAT5 expression profile revealed a detectable band in leaf tissue sample that may be related to its specific roles in this organ.
RNA extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR) Viral RNA was extracted from the collected blood samples by using the High Pure Viral RNA Kit, (Roche Diagnostics, Indianapolis, USA) following the manufacturer
