Abstract :
A highly sensitive and specific reverse transcription polymerase chain reaction enzyme linked immunosorbent assay (RT-PCRELISA)
was developed for the objective detection of nucleoprotein (N) gene of peste des petits ruminants (PPR) virus from
field outbreaks or experimentally infected sheep. Two primers (IndF and Np4) and one probe (Sp3) available or designed for the
amplification/probing of the ‘N’ gene of PPR virus, were chosen for labeling and use in RT-PCR-ELISA based on highest analytical
sensitivity of detection of infective virus or N-gene containing recombinant plasmid, higher nucleotide homology at the primer
binding sites of the ‘N’ gene sequences available and the ability to amplify PPR viral genome from different sources of samples.
RT-PCR was performed with unlabeled IndF and Np4 digoxigenin labeled primers followed by a microplate hybridization probe
reaction with biotin labeled Sp3 probe. RT-PCR-ELISA was found to be 10-fold more sensitive than the conventional RT-PCR
followed by agarose gel based detection of PCR product. Based on the Mean (mean±3S.D.) optical density (OD) values of 47
RT-PCR negative samples, OD values above 0.306 were considered positive in RT-PCR-ELISA. A total of 82 oculo-nasal swabs
and tissue samples from suspected PPR cases were analyzed by RT-PCR and RT-PCR-ELISA, which revealed 54.87 and 58.54%
positivity, respectively. From an experimentally infected sheep, both RT-PCR and RT-PCR-ELISA could detect the virus from 6
days post-infection up to 9 days in oculo-nasal swabs. On post-mortem, PPR viral genome was detected in spleen, lymph node,
lung, heart and liver. The correlation co-efficient between RT-PCR-ELISA OD values and either TCID50 of virus or molecules of
DNA was 0.622 and 0.657, respectively. The advantages of RT-PCR-ELISA over the conventional agarose gel based detection of
RT-PCR products are discussed.
© 2006 Elsevier B.V. All rights reserved.
