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A multiplex TaqMan real time RT-PCR was developed for detection and differentiation of Sweet potato virus G , Sweet potato latent virus and Sweet potato mild mottle virus in one tube. Amplification and detection of a fluorogenic cytochrome oxidase gene was included as an internal control. The assay was compared with a multiplex RT-PCR developed in the initial study for the detection and differentiation of the three viruses and host 18S rRNA. Primers and/or probes of the two assays were designed from conserved regions of each virus. The two assays were optimized for primers/probes and primer concentrations and thermal cycling conditions. Sensitivity and specificity of the assays were compared each other and with other assay. Both assays were evaluated by 74 field samples original from five different provinces of China. Results showed that the TaqMan real time RT-PCR offered rapid, sensitive, effective and reliable for the simultaneous detection and differentiation of the three viruses in sweet potato plants. The assay will be useful to quarantine and certification programs and virus surveys when large numbers of samples are tested.

A generic RT-PCR assay was developed for the universal detection of viruses of the genus Tobamovirus using a novel pair of degenerate primers designed based on conserved regions on replicase genes of 32 tobamoviruses. The assay detected nine tobamoviruses, including six Solanaceae-infecting subgroup tobamoviruses of Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tomato mottle mosaic virus (ToMMV), Tobacco mottle green mosaic virus (TMGMV), Pepper mild mottle virus (PMMoV), Paprika mild mottle virus (PaMMV), one Orchidaceae-infecting tobamovirus of Odontoglossum ringspot virus (ORSV) and two Cucurbitaceae-infecting subgroup tobamoviruses of Cucumber green mottle mosaic virus (CGMMV) and Zucchini green mottle mosaic virus (ZGMMV), with high amplification efficiency, specificity and sensitivity. The assay was applied to detect tobamoviruses in pepper and tomato fields. Five tobamoviruses, PMMoV, TMV, ToMV, ToMMV and TMGMV, were detected from the pepper fields in single and mixed infections. Single infections of PMMoV, ToMV and ToMMV and mix-infection of ToMV + PMMoV were detected from the tomato fields. Among these viruses, PMMoV was first detected from tomato worldwide, while ToMMV was first detected from tomato plants in China. This generic assay is simple, cost-effective and has great potential to detect more tobamoviruses in the field.

Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay was developed for simultaneous detection of the four causal agents of the disease. Four pairs of specific primers based on the conserved regions of each of the four disease agents were used in the one-tube RT-PCR. The RT-PCR products consisted of fragments of 1049 base pairs (bp) for TBTV, 792bp for TVDVaRNA, 598bp for Sat-TBTV and 357bp for TVDV, and their origins were confirmed by sequencing. Primer concentrations and cycling condition were optimized for the multiplex RT-PCR. The detection limit of the assay was up to 10(-4) dilution. The assay was evaluated using tobacco plants infected naturally with one to four target viruses, transmission vector of aphids and field samples collected from Yunnan, Hunan, and Guizhou province, China. The results show that the multiplex RT-PCR is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in tobacco and aphid. This assay will be useful for virus surveys when large numbers of samples are tested.

A simple and sensitive dot blot hybridization assay using a digoxigenin-labeled cRNA polyprobe was developed for the simultaneous detection of six viroids that infect pome and stone fruit trees. The polyprobe was constructed by cloning sequentially partial sequences of each viroid into a single vector, with run-off transcription driven by the T7 promoter. All six viroids were detectable within a dilution range of 5−3 to 5−4 in total nucleic acid extracts from infected trees. Individual trees were co-inoculated to create mixed infections and all four pome fruit viroids and both stone fruit viroids could be detected in pear and peach trees, respectively, using the polyprobe. The results of the assays using the polyprobe were comparable to those using single probes. The methods were validated by testing geographically diverse isolates of viroids, as well as field samples from several collections in the US. The assay offers a rapid, reliable and cost-effective approach to the simultaneous detection of six fruit trees viroids and has the potential for routine use in quarantine, certification, and plant genebank programs where many samples are tested and distributed worldwide.

Sugarcane mosaic disease (SMD) caused by the Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV) and sugarcane yellow leaf disease (SYLD) caused by the Sugarcane yellow leaf virus (SCYLV) are the two most prevalent and economically important viral diseases of sugarcane. In this study, a one-step quadruplex reverse transcription (RT)-PCR method that employed virus-specific primers was developed for the simultaneous detection and differentiation of SCMV, SrMV, SCSMV and SCYLV. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by simplex and quadruplex RT-PCR. The optimum primer combinations and concentrations, RT temperature and time, and PCR annealing temperature and extension time were determined for the quadruplex RT-PCR. The assay was then validated using sugarcane samples affected with SMD and/or SYLD collected from sugarcane breeding fields and farmers' fields in southern China.

A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

A one-step reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed to detect Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV) in woody indicators and naturally infected Prunus spp. Viral RNA suitable for RT-PCR was obtained by a simple trapping method that did not require either extraction of double-stranded RNA (dsRNA) or total RNA, availability of virus antibodies, or purification of viral particles. Consensus primers, degenerate primers and virus-specific primers, whose designs were based on alignments of available cherry flexivirus sequences, were tested to amplify viral genomic fragments of six CGRMV isolates and one CNRMV isolate. RT-PCR allowed CGRMV detection in total RNA and viral RNA preparations equivalent to 400mug and 4mug of infected leaf tissue, respectively. CGRMV was detected in tender shoots, leaves, bark and root tips, and the strongest bands were obtained using young leaves. Detection was less consistent in summer when the temperature was elevated and plant tissues were old. A direct comparison of the RT-PCR and grafting assays indicated that the RT-PCR assay is sensitive, rapid and reliable. The method will improve the routine diagnosis of cherry flexiviruses in Prunus spp.

Four closely related potyviruses, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG) and/or Sweet potato virus 2 (SPV2), are involved in sweet potato virus disease complexes worldwide. Identification and detection of these viruses are complicated by high similarity among their genomic sequences, frequent occurrence as mixed infections and low titer in many sweet potato cultivars. A one-tube multiplex reverse transcription-PCR (mRT-PCR) assay was developed for simultaneous detection and differentiation of SPFMV, SPVC, SPVG and SPV2. Four specific forward primers unique to each virus and one reverse primer based on the region conserved in all four viruses were selected and used in the assay. The mRT-PCR assay was optimized for primer concentration and cycling conditions. It was tested using sweet potato plants infected naturally with one to four target viruses and then evaluated using field samples collected from southwestern China. The mRT-PCR assay is reliable and sensitive as a simple, rapid and cost-effective method to detect these pathogens in sweet potato. This assay will be useful to quarantine and certification programs and virus surveys when large numbers of samples are tested.