Development and comparison of genome detection assays for the diagnosis of foot-and-mouth disease suspected clinical samples.
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.
Mohapatra
Jajati Keshari
JK
Project Directorate on Foot-and-Mouth Disease, Indian Veterinary Research Institute Campus, Mukteswar-Kumaon, Nainital 263138, Uttaranchal, India.
Sanyal
Aniket
A
Hemadri
Divakar
D
Tosh
Chakradhar
C
Palani
Giridharan
G
Rasool
Thaha Jamal
TJ
Bandyopadhyay
Santanu Kumar
SK
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
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