| Potato is a staple food crop and is widely grown in China. One of the important factors affecting potato quantity and quality is the diseases caused by several potato viruses. Production of virus or viroid free seed-potato using tissue culture is the main measure of controlling potato virus diseases. In producing virus free potato seeds, it is a priority to develop a rapid, sensitive and accurate detection method for products inspection. Therefore, the main purpose in the present study is to test and establish a technique using the multiplex reverse transcription polymerase chain reaction (m-RT-PCR) to detect several viruses simultaneously based on rapid sample preparation and optimal detection system as well as solid pre-coated reagent potato virus and viroid detection kit.1. Design of the primers for potato viruses: The primer pairs for PVX, PVY, PLRV and PVS were designed based on the coat protein region, the primers for PVA based on the P1 gene region, the primers for PSTVd based on the entire genome. One step or two step m-RT-PCR system was developed for the simultaneous detection of five potato viruses and viroid; the amplification fragments of 562bp (PVX), 480bp (PVY), 336bp (PLRV), 182bp (PVS), 225bp (PVA), 360bp (PSTVd) for different potato viruses and viroid were generated respectively.2. Establishment of rapid viral ssRNA preparation method: The main component of virus ssRNA soaking-solution included active ionic surfactants (0.5% Triton X-100 R) and polyphenol oxydase inhibitors (0.4% Na2SO3), which can rapidly release the potato virus ssRNA from plant tissues. The extracting method was rapid and simple, suitable for extracting ssRNA of PVY, PVX, PLRV, PVA, PVS and PSTVd from potato tissues including leaf, petiole, stem and tuber. First, tissue of infected potato leaves (5mg~ 10mg), petioles or stem (20mg~30mg), tubers (30mg~40mg) was added into 100 ul RNA soaking-solution and incubated 20 min at 37 in a microwave; the suspension from the liquid was collected after centrifugation and was directly used for cDNA synthesizing in RT-PRC. The suspensions of virus or viroid extracts mixed with macromolecular stabilizer can be stored for up to three months and are still good for simplex, multiplex RT-PCR.3.Development of two-step RT-PCR system: The two steps simplex, duplex, multiplexRT-PCR developed could be used to detect multiple potato viruses simultaneously. The reverse transcription mixture of simplex RT-PCR contained 1XRT reaction buffer (including 3 mmol/L MgCl2), 2.5U RNasin, 200U MMLV reverse transcriptase, 0.5 mmol/L dNTPs, 0.5 w mol/L anti-sense primer. Reverse transcription samples (10 w 1) cDNA were incubated at 25℃ for 20 min, transferred to 42℃ for 30 min for RT and the reaction was stopped by heating to 95℃ for 2 min. The final concentrations of the remaining reagents for the PCR were: 1× PCR buffer, 0.2 mmol/L dNTPs, 1U Amplitaq DNA polymerase, 2.5 mmol/L MgCl2, antisense and sense primers each, 0.5u mol/L. PCR samples (25 ul) were amplified in 30 cycles. A cycle consisted of 30s each of denaturation (94℃), annealing (60℃), extension (72℃) and final extension of 10min (72℃). For duplex RT-PCR, the concentration of dNTPs was 1.0 mmol/L in RT mixture; the others were the same as those in the simplex RT-PCR. For multiplex RT-PCR, the RT mixture contained 1.5 mmol/L dNTPs, 10U RNasin, 100U MMLV- reverse transcriptase; the PCR mixture contained 3.5 mmol/L MgCl2, 2 U AmpliTaq DNA polymerase, and 0.5 mmol/L dNTPs. The sample was transferred to 42℃ for 30min for RT state, 72℃ for extension for 1min for PCR, the others were similar to simplex RT-PCR.4. Set up of one step RT-PCR system: One-step RT-PCR developed could detect several potato viruses simultaneously and the reaction buffer was suitable for RT and PCR assays. The simplex RT-PCR reaction reagent contained 1×reaction buffer, 5 U RNasin, 50U MMLV-reverse transcriptase, 0.5 mmol/L dNTPs, 1U Amplitaq DNA polymerase, 1.5 mmol/L MgCl2, anti-sense and sense primer each 0.5 umol/L. For duplex RT-PCR, 2.5 mmol/L MgCl2 an...
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