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 using organic solvents or Fast DNA kit. SSV cDNA was amplified using viral specific primers and the amplified SSV cDNA (amplicon) was DIG-labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system by a microtiter plate using a biotinylated cDNA (22 nt), cDNA (789 nt) or cRNA (789 nt) capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is 10-100 fold more sensitive than agarose gel electrophoretic analysis 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. Complete nucleotide sequence of SSV from Naga Hammady, Egypt, revealed that SSV-EG is a new species of SSV that shares 66% nucleotide identity with the virus species from Natal, South Africa.
Shamloul
A M
AM
Fruit Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA. mshamloul@dir.nidcr.nih.gov
Abdallah
N A
NA
Madkour
M A
MA
Hadidi
A
A
D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D004534:Egypt; D004797:Enzyme-Linked Immunosorbent Assay; D017791:Geminiviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016679:Genome, Viral; D008969:Molecular Sequence Data; D010944:Plants / Q000821:virology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral