Development of a N gene-based PCR-ELISA for detection of Peste-des-petits-ruminants virus in clinical samples.
A highly sensitive N gene-based PCR-ELISA for the detection of Peste-des-petits-ruminants virus (PPRV) was developed. The RT-PCR yielded a digoxigenin (DIG)-labeled product of 336 bp comprising a sequence from PPRV N gene, which was then detected by ELISA. The assay could detect the viral RNA in PPRV-infected tissue culture fluids with a titer as low as 0.1 TCID(50)/ml. The assay is 10,000 times more sensitive than a classical RT-PCR combined with agarose gel electrophoresis. The assay could detect the virus in the clinical samples, which were negative by conventional sandwich ELISA (S-ELISA). The percentage positivity of the assay in detecting the virus in clinical samples was 66.2% compared to 48.6% for S-ELISA. The assay was more sensitive than S-ELISA also in detecting the virus in early as well as late phases of the disease. In addition, the assay could also be used for differential diagnosis of PPRV and Rinderpest virus (RPV).
Saravanan
P
P
Division of Virology, Indian Veterinary Research Institute, Mukteswar-Kumaon, Nainital, Uttaranchal, 263 138, India. drsaravana72@dr.com
Singh
R P
RP
Balamurugan
V
V
Dhar
P
P
Sreenivasa
B P
BP
Muthuchelvan
D
D
Sen
A
A
Aleyas
A G
AG
Singh
R K
RK
Bandyopadhyay
S K
SK
D000818:Animals; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006041:Goats / Q000821:virology*; D009693:Nucleic Acid Hybridization; D018107:Peste-des-petits-ruminants virus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014709:Vero Cells