A new multiplex RT-PCR assay for serotyping of bluetongue virus

Abstract

Among all viral diseases affecting small ruminants, bluetongue is the one that affects adversely to an alarming extent. The current available diagnosis/serotyping of bluetongue virus (BTV) is time consuming, costly, and requires to screen individually for each of the 29 distinct serotypes. The present study was conducted with the objective of developing a multiplex reverse transcription PCR (mRT-PCR) assay for serotyping of BTV, especially for serotypes BTV-1, 2, 9, 12, 16, 21 and 23 predominantly circulating in India. The type specific primers for the selected BTV serotypes were designed targeting the serotype specific segment-2 region of BTV based on the reference serotype sequences of Indian isolates available in GenBank. The mRT-PCR was conducted in two groups - group A for BTV-1, 9, 12, 21 and group B for BTV-2, 16 and 23. A panel of 25 BT suspected clinical samples were typed by mRT-PCR. The results were further validated by the gold standard serum neutralization test (SNT). A seroprevalence of 60% for BTV- 2, 10% for BTV- 9, 15% for BTV- 1, 10% for BTV- 16 and 5% for BTV- 23 were observed. Further, we noticed that there was a mixed serotype infection in 10% of BTV positive samples. In conclusion, we report the development of a novel mRT-PCR assay for a rapid and cost-effective nucleic acid based serotyping of BTV having the specificity same as SNT.