title: Development and comparison of genome detection assays for the diagnosis of foot-and-mouth disease suspected clinical samples.
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.
authors.affiliation: Project Directorate on Foot-and-Mouth Disease, Indian Veterinary Research Institute Campus, Mukteswar-Kumaon, Nainital 263138, Uttaranchal, India.
authors.forename: Jajati Keshari
authors.initials: JK
authors.lastname: Mohapatra
authors.forename: Aniket
authors.initials: A
authors.lastname: Sanyal
authors.forename: Divakar
authors.initials: D
authors.lastname: Hemadri
authors.forename: Chakradhar
authors.initials: C
authors.lastname: Tosh
authors.forename: Giridharan
authors.initials: G
authors.lastname: Palani
authors.forename: Thaha Jamal
authors.initials: TJ
authors.lastname: Rasool
authors.forename: Santanu Kumar
authors.initials: SK
authors.lastname: Bandyopadhyay
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D003124:Colorimetry; D003546:Cysteine Endopeptidases / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005536:Foot-and-Mouth Disease / Q000175:diagnosis* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
chemical_list: D000956:Antigens, Viral; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C103557:virus-infection-associated antigen, Foot-and-mouth disease virus; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases