Journal of Virological Methods 137 (2006) 14–20 Development and comparison of genome detection assays for the diagnosis of foot-and-mouth disease suspected clinical samples Jajati Keshari Mohapatra, Aniket Sanyal ∗ , Divakar Hemadri, Chakradhar Tosh, Giridharan Palani, Thaha Jamal Rasool, Santanu Kumar Bandyopadhyay 1 Project Directorate on Foot-and-Mouth Disease, Indian Veterinary Research Institute Campus, Mukteswar-Kumaon, Nainital 263138, Uttaranchal, India Received 23 March 2006; received in revised form 22 May 2006; accepted 25 May 2006 Available online 30 June 2006 Abstract Detection of foot-and-mouth disease virus (FMDV) from clinical specimens by conventional sandwich enzyme-linked immunosorbent assay (ELISA) and virus isolation in cell culture is often compromised owing to limited sensitivity and inactivation during transit, respectively. A RT-PCR (oligoprobing) ELISA in both solid and aqueous phase hybridization formats targeting an across serotype conserved site at 3C–3D region was developed and its effectiveness was compared with that of the known targets at the IRES region. A non-isotopic RNA dot hybridization assay with colorimetric detection targeting both the IRES and the 3D region were also validated, which is capable of handling high throughput samples with ease. RT-PCR (oligoprobing) ELISA and dot hybridization assay showed 1000- and 10-fold greater sensitivity than the sandwich ELISA, respectively. Robustness of these diagnostic methods was explored by examining on sandwich ELISA-negative clinical samples. Both the assays developed in the present study were able to detect viral genomes in samples undetectable by conventional ELISA, thereby demonstrating ‘proof of sensitivity’. Although the potential of these assays for providing definitive diagnosis in carrier hosts and in species where clinical disease is inapparent remains to be examined, nevertheless these assays can be adapted for comprehensive surveillance of foot-and-mouth disease in India. © 2006 Elsevier B.V. All rights reserved. Keywords: FMD virus; Genome detection; RT-PCR ELISA; Dot hybridization 1. Introduction Foot-and-mouth disease (FMD) is endemic in India and out- breaks are reported due to serotypes O, A and Asia1 round the year. Serotype C has not been reported since 1995. FMD is an Office International des Epizooties list A disease and is considered to be the most contagious infectious agent of domestic animals. Traditionally, routine laboratory diagnosis of FMD is obtained by subjecting the suspected epithelial sam- ples to enzyme-linked immunosorbent assay (ELISA), accom- panied by cell culture isolation of the virus (Alexandersen et al., 2003). Clinical diagnosis of FMD has always been compli- cated: sandwich ELISA, although simple, rapid and can handle ∗ Corresponding author. Tel.: +91 5942 286004; fax: +91 5942 286307. E-mail addresses: asanyal68@rediffmail.com, asanyal68@email2me.net (A. Sanyal). 1 Present address: Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, Krishi Bhavan, 110001 New Delhi, India. large number of samples, suffers from low sensitivity. As a result a significant percentage (20–30%) of FMD suspected sam- ples referred to Project Directorate on FMD (PD FMD), India, from all over the country remain undiagnosed (Giridharan et al., 2005). Although virus isolation is considered to be the “gold standard”, it is slow, cumbersome, labour-intensive and fails on occasions where samples have to withstand fluctuation in tem- perature and pH during transit and other conditions (Reid et al., 1998). Diagnosis based on detection of specific antibody may lead to false-positive result in the case of possible pre- vious infection in an endemic zone and countries employing vaccination as a control measure. Clinical diagnosis in sheep and goats is complicated by the fact that these species do not show overt symptoms (Callens et al., 1998). Furthermore, sev- eral other vesicular diseases causing viral infections like swine vesicular disease cannot be distinguished from FMD by clinical symptoms alone (Nunez et al., 1998; Lomakina et al., 2004). Establishment of persistent infection in unvaccinated as well as vaccinated animals, particularly cattle (Burrows, 1966; Salt et 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.05.022