Molecular detection of a fragment of bluetongue virus in sheep from different regions of Mexico

Abstract

Bluetongue disease (BTD) affects various species of wild and domestic ruminants. In Mexico, the disease, caused by the bluetongue virus (BTV) is still regarded as exotic, despite the fact that antibodies have been detected on several occasions. The objective was to establish molecular techniques using a synthetic gene, including the genes NS1 and NS3 as positive controls for the diagnosis of BTV in samples of sheep from different regions of the country. A total of 320 total whole blood samples were obtained from sheep. The samples obtained were evaluated by end-point RT-PCR and real-time RT-PCR, the conditions having been established by the work group. Twelve sheep samples were found to be positive for the detection of NS1; these samples were sequenced, and a fragment of 101 base pairs was obtained. Upon alignment, were obtained identities with sequences reported in GenBank with NS1 fragments ranging from 89 % (p= 1e-12) to 98 % (p= 4e-13), corresponding to serotypes 10, 11 and 12. From these samples, two positive sheep samples were obtained using real-time PCR (RT-PCR): one from Chiapas (Chiapas breed), and the other, from Tamaulipas (Suffolk breed). The results of the RT-PCR were corroborated by CPA-SENASICA. This work provides evidence, for the first time in Mexico, of the importance of using a synthetic gene as a positive control to perform BSL-2 detection in official laboratories, which in a health emergency is of utmost importance.