Sensitive detection of the Egyptian species of sugarcane streak virus by PCR-probe capture hybridization (PCR-ELISA) and its complete nucleotide sequence

A rapid and sensitive assay for the specific detection of Sugarcane streak virus (SSV) using PCR-probe capture hybridization (PCR-ELISA) was developed . Nucleic acids suitable for PCR were extracted from SSV-infected tissue us ing organic solvents or Fast DNA kit. SSV cDNA was amplified using viral sp ecific primers and the amplified SSV cDNA (amplicon) was DIG-labelled durin g the amplification process. The amplicon was then detected in a colorimetr ic hybridization system by a microtiter plate using a biotinylated cDNA (22 nt), cDNA (789 nt) or cRNA (789 nt) capture probe. This system combines th e specificity of molecular hybridization, the ease of the colorimetric prot ocol, and is 10-100 fold more sensitive than agarose gel electrophoretic an alysis in detecting the amplified product. Long cDNA or cRNA capture probe was 2-7 fold more sensitive than the oligo cDNA probe for the detection. Co mplete nucleotide sequence of SSV from Naga Hammady, Egypt, revealed that S SV-EG is a new species of SSV that shares 66% nucleotide identity with the virus species from Natal, South Africa. (C) 2001 Elsevier Science B.V. All rights reserved.