Study Of Molecular Diagnositic Methods For Detection Of Potato Viruses And A Viroid

More than 25 viruses have been reported to naturally infect potato crops. Of them, some cause severe damage to the crop, which potentially decrease yield and quality of potato. As reported, viruses producing acute damage to the crop in native potato-producing areas have Potato virus A (PVA), Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS), Potato leaf roll virus (PLRV) and Potato spindle tuber viroid (PSTVd). In this study, four sets of methods were developed for different requirements of detection of potato viruses according to numbers of potato viruses and test samples. The first method is virion-direct absorbent-reverse transcriptase-polymerase chain reation (VD A-RT-PCR), designed for detection of few viruses in some samples; the second is multiplex RT-PCR combined with 18S rRNA as an internal control, designed for simultaneous detection of five important viruses above mentioned; the third is oligonucleotide microarray technique, designed for simultaneous detection of seven potato viruses and one viroid; the last one is glass slide nucleic acid hybridization, designed for detecting potato viruses or viroid from large numbers of potato samples in one reaction.First, cloning and sequencing for the conserved sequences of PVA, PVX, PVY, PVS, PLRV, tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), PSTVd and 18S rRNA were perfomed according to molecular cloning method. Results of homology analyses showed that, the PVX isolate had a similarity of 94.0%-100.0% to group II strains, and 83.0%-86.0% to group I strains. The isolate should be classified to group II. The CMV isolate was 94.5%-98.6% identical to subgroup I strains, and 75.7%-78.9% identical to subgroup II strains. The isolate should be a strain of subgroup I. However, PVY did not have association between sequence homologies and strains. Other viruses or PSTVd had a similarity of up to 94.0% with few exceptions. These high homogies suggested that these sequences could be used to prepare virus-specific or strain-specfic probes for nucleic acid hybridization.Virion-direct absorbent-reverse transcriptase-polymerase chain reaction (VDA-RT-PCR). This method was designed for detecting single virus from some potato samples. We attempted to utilize the character of non-specific absorption between test tube and viral particles to establish a rapid, simple, phenol-chloroform-free method for preparation of potato viral RNAs. This method was named 'virion-direct absorbent'. VDA combined with RT-PCR (VDA-RT-PCR) was used for detection of potato viruses. Results from a series of conditional assays showed that, optimal protocol was set up by utilizing general extraction buffer, incubation for one hour, and denaturing viral particles at 70°C. Absorbed virions could be preserved at room temperature for up to 11 days. The optimal VDA-RT-PCR protocol could detect 1.25 ng PVX, similarly sensitive to ELISA method. Although the protocol had not high sensitivity, it was very simple, timesaving and costsaving. Viral RNA could be prepared in only one tube.Multiplex RT-PCR. This method combined with 18S rRNA as an internal control, was designed for simultaneous detection of five important viruses. Some reports used a plant NADH dehydrogenase mRNA as an internal control for the success of RNA extraction and RT-PCR reaction. However, 18S ribosomal RNA (rRNA) usually be used as an internal control in real-time RT-PCR systems. Therefore, in order to search for a perfect internal control, the 18S rRNA was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection limit of 18S rRNA was 5 magnitudes higher than that of nad2 mRNA, demonstrating that 18S rRNA used as an internal control was more sensitive. The 18S rRNA also displayed degradation kinetics more similar to that of PVX. Thus, 18S rRNA was used as an internal control in a multiplex RT-PCR system for simultaneous detection of five economically important potato viruses. By a series of optimization assays, optimal multiplex RT-PCR was achieved by increasing concentrations of Taq HS DNA polymerase and MgCl₂ to be 0.1 U/μl and 4.0 mM, respectively. The optimal multiplex RT-PCR system could simultaneously amplify cDNAs from PVA, PVX, PVY, PLRV, PVS, and 18S rRNA, and was able to detect all viruses under different combinations of the viruses. The technique was 100- fold greater for detection of PVX than that of commercial DAS-ELISA, and also could detect PVX and PVS in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.Oligonucleotide microarray. This method was designed for simultaneous detection of seven potato viruses and one viroid. Recently, DNA microarray technique has been applied to detection of potato viruses. Use of reverse transcription labeling method made these DNA microarray systems had lower sensitivities, which was the major drawback of these systems. Hence, we attempted to establish an oligonucleotide microarray of high sensitivity and specificity for simultaneous detection of seven potato viruses and potato spindle tuber viroid (PSTVd), using multiplex labeling PCR method and about 25mer oligonucleotides. Results showed that absence of cross hybridization occurred between labeled DNAs and heterogeneous probes, indicating that the oligonucleotide microarray established in this study was of high specificity. Through optimization of two multiplex PCR reactions, PLRV, PVY, PVX, PVA, PVS, CMV, TMV, PSTVd, and 18S rRNA could be simultaneously labeled. Combined with the multiplex labeling PCR method, the oligonucleotide microarray could detect CMV in the 10⁹ fold dilution of CMV RNA; however, the detection limit of nucleic acid spot hybridization technique was 10⁵ or 10⁶ fold dilution. Obviously, use of multiplex labeling PCR method significantly increased detection sensitivity of DNA microarray. The sensitivity of the oligonucleotide DNA microarray presented in this study was much higher than those of other DNA microarrays of detecting potato viruses reported by other researchers.Glass slide hybridization. This method was designed for detecting potato viruses or viroid from large numbers of potato samples in one reaction. As a diagnostic tool, DNA microarray has unique merit of simultaneous detection of multiple pathogens in one reaction. However, this technique is not competent for detecting few viruses in large numbers of potato samples. To address this problem, an attempt was made to establish a set of modified dot blot hybridization system, by replacement of ³²P-labeled probes with CY5-labeled probes and nylon membrane with aminosilane-coated glass slide as a RNA supporter. Results from conditional assays showed that formamide and DMSO spotting solutions used for nylon membrane were not suitable for aminosilane-coated glass slide; RNA dissolved in the two solutions showed lower binding efficiency than RNA dissolved in SSC solution. SSC (5×) of high concentration was beneficial to RNA binding on surface of aminosilane-coated glass slides; however, relatively low concentration (1×) showed obvious linear relativity. Various types of glass slides had different capabilities of binding RNA each other. Aminosilane-coated glass slides produced much higher hybridization signals than poly-L-lysine-coated slides and aldehyde-coated slides. Hence, sensitivity of the glass slide hybridization was determined using 1×SSC as spotting solution and aminosilane-coated glass slides as a RNA supporter. Probes prepared by PCR method and random labeling method could detect as little as 1.71 pg and 13.67 pg TMV RNA, respectively; nylon membrane hybridization with ³²P-labeled probe could detect 6.83 pg TMV RNA. Although faint cross-hybridization occurred between PSTVd-specific probe and total RNA sample isolated from PVY-infected Nicotiana glutinosa leaf tissue, absence of cross-hybridization aroused among three Potyvirus species, including PVA, PVY, and Zucchini yellow mosaic virus (ZYMV), demonstrating that the established method possessed high specificity. A test of field-grown potato samples suggested that the method was capable of detecting potato virus(es) in a great number of potato samples.Every diagnostic method has its advantages and disadvantages, including the four sets of methods established in this study. For example, it will increase expense that use multiplex RT-PCR to detect single virus; use of VDA-RT-PCR to do will save expense (especial reagents for extraction of nucleic acid), time and labor. If few viruses will be detected in large numbers of potato samples, we should choose glass slide nucleic acid hybridization, instead of oligonucleotide microarray method. If many viruses will be detected in few samples, we should select multiplex RT-PCR or oligonucleotide microarray, rather than glass slide nucleic acid hybridization. Therefore, we can pertinently select suitable diagnostic methods to decrease expense, time and labor as little as possible, according to different detecting requirements.