Abstract
Bluetongue is an insect-borne disease of domestic and wild ruminants that requires strict monitoring by sensitive, reproducible and robust methods. Real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis has become the method of choice for routine viral diagnosis. As false-negative test results can have serious implications; an internal/external control system should be incorporated in each analysis to detect RT-qPCR failure due to poor sample quality, improper nucleic acid extraction and/or PCR inhibition. To increase the diagnostic capacity and reduce costs, it is recommended to use a multiplex strategy which enables the amplification of multiple targets in a single reaction. This chapter describes the application of a triplex RT-qPCR for the simultaneous detection of bluetongue viral RNA, an internal control and an external control. The primer and probe sequences of the BTV RT-qPCR were taken from Toussaint et al. (J Virol Methods 140:115–123, 2007), whereas the internal and external RT-qPCRs were specifically designed to detect endogenous glyceraldehyde-3-phosphate dehydrogenase mRNA and a synthetic RNA, respectively. To maximize the sensitivity of the assay, the primer concentrations of the internal/external control reactions were limited and the amount of Taq DNA polymerase was increased. A comparison of the singleplex versus triplex RT-qPCR indicated that the triplex RT-qPCR exhibits a higher analytical sensitivity. Due to the incorporation of the internal/external control system, the triplex RT-qPCR allows an even more reliable and rapid diagnosis of bluetongue than the previously described singleplex RT-qPCR (J Virol Methods 140:115–123, 2007).
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References
MacLachlan NJ (1994) The pathogenesis and immunology of bluetongue virus infection of ruminants. Comp Immunol Microbiol Infect Dis 17:197-206
Verwoerd DE (2004) Bluetongue. In: Tustin RC, Coetzer JA (eds) Infectious diseases of livestock. Oxford University Press, Cape Town, pp 1201-1220
Mertens PP, Diprose J, Maan S, Singh KP, Attoui H, Samuel AR (2004) Bluetongue virus replication, molecular and structural biology. Vet Ital 40:426-437
MacLachlan NJ, Osburn BI (2006) Impact of bluetongue virus infection on the international movement and trade of ruminants. J Am Vet Med Assoc 228:1346-1349
Rosenstraus M, Wang Z, Chang S-Y, DeBonville D, Spadoro JP (1998) An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol 36:191-197
Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P (2004) Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 42:1863-1868
Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JDC, Wengenack NL, Rosenblatt JE, Cockerill FR III, Smith TF (2006) Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19:165-256
Chamberlain JS, Gibbs RA, Ranier JN, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucl Acids Res 16:11141-11156
Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE (2000) Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 13:559-570
Vandenbussche F, Vanbinst T, Vandemeulebroucke E, Goris N, Sailleau C, Zientara S, De Clercq K (2008) Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR. J Virol Methods 152:13-27
Toussaint JF, Sailleau C, Breard E, Zientara S, De Clercq K (2007) Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J Virol Methods 140:115-123
Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of thermus aquaticus DNA polymerase. Proc Natl Acad Sci 88:7276-7280
Acknowledgments
The study was supported by grants from DG4-6406 Bluetongue-RF 6187; the Belgian Federal Agency for the Safety of the Food Chain; EC-BTVAC SSPE-CT-2006-044211; EC-MedReoNet-SSPE-CT-2006-04428; EC-EPIZONE FOOD-CT-2006-016236 and the Veterinary and Agrochemical Research Centre. We wish to thank all technicians of the VAR who contributed to this study.
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Vandenbussche, F., Vandemeulebroucke, E., De Clercq, K. (2010). Simultaneous Detection of Bluetongue Virus RNA, Internal Control GAPDH mRNA, and External Control Synthetic RNA by Multiplex Real-Time PCR. In: King, N. (eds) RT-PCR Protocols. Methods in Molecular Biology, vol 630. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-629-0_7
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DOI: https://doi.org/10.1007/978-1-60761-629-0_7
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