Sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization.

A rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction (RT-PCR) -probe capture hybridization (RT-PCR-enzyme-linked immunosorbent assay (ELISA)) was developed. The assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. Clarified sap extract from infected leaf tissue was treated first with GeneReleaser polymeric matrix to remove inhibitors of RT-PCR reactions. Viroid cDNA was then synthesized and amplified using viroid specific primers in RT-PCR assays and the amplified viroid cDNA (amplicon) was digoxigenin (DIG) -labelled during the amplification process. The amplicon was then detected in a colorimetric hybridization system in a microtiter plate using a biotinylated cDNA capture probe. This system combines the specificity of molecular hybridization, the ease of the colorimetric protocol, and is at least 100-fold more sensitive than gel electrophoretic analysis in detecting the amplified product. Viroid cRNA may replace viroid cDNA as the capture probe. The cRNA probe was several fold more sensitive than the cDNA probe for viroid detection. Six to seven hours are needed to complete the RT-PCR-ELISA for viroid detection from infected leaf tissue.

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

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.

Occurrence of Peach Latent Mosaic Viroid in Stone Fruits and Its Transmission with Contaminated Blades.

Peach latent mosaic viroid (PLMVd) is widely distributed (approximately 55%) in peach germplasm from Europe, Asia, North America, and South America. PLMVd, or a closely related viroid, was occasionally detected in cherry, plum, and apricot germplasm from countries in Europe or Asia. The cherry isolate of PLMVd is 337 nucleotides in length and is 91 to 92% homologous to PLMVd isolates from peach. Molecular hybridization experiments demonstrated that PLMVd is not related to the agent of peach mosaic disease. PLMVd was readily transmitted (50 to 70%) by contaminated blades to green shoots and lignified stems of peach GF-305 plants. These results indicate that the viroid may be transmitted in orchards with contaminated pruning equipment.

Apricot latent virus: a new species in the genus Foveavirus.

Extraction of viral double-stranded RNA from peach leaves infected with Apricot latent virus (ALV) followed by molecular cloning of synthesized cDNA and its sequencing, suggested that ALV is a new virus, whose coat protein (CP) coding region contains Apple stem pitting virus (ASPV)-related sequences. The sequenced portion of the ALV genome (1,444 nt) includes the putative CP gene and the 3' non-translated region. The 5' portion of this fragment (1-651 nt) is highly distinct whereas the 3' portion is 77% identical to the corresponding region of ASPV. Molecular hybridization experiments using a cRNA probe to ASPV with ALV-infected leaf tissue extracts also revealed that the genome of ALV contains nucleotide sequences related to that of ASPV. Western blots of tissue extracts indicated that ALV coat protein reacted with polyclonal antiserum against ASPV; however, the ALV CP differs in size from that of ASPV. ALV was graft-transmitted to several Prunus rootstocks. Based on the available sequence data, serological observations and bioassays we propose that ALV is a new species in the genus Foveavirus, typified by ASPV. ALV-specific PCR-primers and viral-specific cRNA probes developed in this investigation may be useful for detecting the virus and for studying its epidemiology and geographical distribution.