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Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus.

ABSTRACT: A novel two-step, SYBR Green I based real-time RT-PCR assay was developed for detection and quantification of BCoV using ABI PRISM 7500 sequence detection system. The assay was carried out using two sets of primers designed to amplify highly conserved sequences of the nucleocapsid gene of BCoV and the internal control, bovine glyceraldehyde-3-phosphate dehydrogenase, RNA. Specific identification of both targets was elucidated by melt curve analysis, in which the BCoV amplified product generated a melt peak at 78.35 ± 0.26 °C and the internal control RNA at 82.54 ± 0.32 °C. The assay was highly specific since all negative controls and other viruses of clinical and structural relevance failed to develop any positive results. The detection limit of the reaction was 10(3) plasmid copies and 1.17 × 10(-3) TCID(50) of the tissue culture propagated virus. Standard deviation and coefficient of variation was low for both intra-assay and inter-assay variability. The assay performance on field samples was evaluated on 103 (68 fecal and 35 nasal) swab specimens and compared with the conventional RT-PCR assay. The results of both assays matched for the diagnosis of 65 fecal and 33 nasal samples. However, three fecal and two nasal samples tested negative in gel-based assay were positive for the real-time RT-PCR. The robustness and a high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and in BCoV research.

SUBMITTER: Amer HM

PROVIDER: S-EPMC7127634 | biostudies-literature | 2011 Apr-Jun

REPOSITORIES: biostudies-literature

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Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus.

Amer Haitham M HM Almajhdi Fahad N FN

Molecular and cellular probes 20110317 2-3

A novel two-step, SYBR Green I based real-time RT-PCR assay was developed for detection and quantification of BCoV using ABI PRISM 7500 sequence detection system. The assay was carried out using two sets of primers designed to amplify highly conserved sequences of the nucleocapsid gene of BCoV and the internal control, bovine glyceraldehyde-3-phosphate dehydrogenase, RNA. Specific identification of both targets was elucidated by melt curve analysis, in which the BCoV amplified product generated ...[more]

PMID: 21419844

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Project description:BackgroundToday, when more than 60% of animal diseases are zoonotic, understanding their origin and development and identifying protective immune responses in ruminants are major challenges. Robust, efficient and cost-effective tools are preconditions to solve these challenges. Cytokines play a key role in the main mechanisms by which the immune system is balanced in response to infectious pathogens. The cytokine balance has thus become the focus of research to characterize immune response in ruminants. Currently, SYBR Green reverse transcriptase quantitative PCR (RT-qPCR) is the most widely method used to investigate cytokine gene expression in ruminants, but the conditions in which the many assays are carried out vary considerably and need to be properly evaluated. Accordingly, the quantification of gene expression by RT-qPCR requires normalization by multiple reference genes. The objective of the present study was thus to develop an RT-qPCR assay to simultaneously quantify the expression of several cytokines and reference genes in three ruminant species. In this paper, we detail each stage of the experimental protocol, check validation parameters and report assay performances, following MIQE guidelines.ResultsTen novel primer sets were designed to quantify five cytokine genes (IL-4, IL-10, IL-12B, IFN-γ and TNF-α) and five reference genes (ACTB, GAPDH, H3F3A, PPIA and YWHAZ) in cattle, sheep, and goats. All the primer sets were designed to span exon-exon boundaries and use the same hybridization temperature. Each stage of the RT-qPCR method was detailed; their specificity and efficiency checked, proved and are reported here, demonstrating the reproducibility of our method, which is capable of detecting low levels of cytokine mRNA up to one copy whatever the species. Finally, we checked the stability of candidate reference gene expression, performed absolute quantification of cytokine and reference gene mRNA in whole blood samples and relative expression of cytokine mRNA in stimulated PBMC samples.ConclusionsWe have developed a novel RT-qPCR assay for the simultaneous relative quantification of five major cytokines in cattle, sheep and goats, and their accurate normalization by five reference genes. This accurate and easily reproducible tool can be used to investigate ruminant immune responses and is widely accessible to the veterinary research community.

Dataset Information

Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus.

Publications

Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus.

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