| Potato（Solanum tuberosum L.） is the fourth food crops in the world.China is the biggist potato production country in the world,with potato planting area of 4.9015 million ha and production of 70.338 million t in 2006.However,the potato yield of China（14.35t / ha） was approximate to the world level which was 16.74t / ha and much lower than that of many developed countries（34.415-45.33 t / ha）.Although it plays a significant and vital role in agriculture and industry,the potato is subject to many diseases and pests, among which the virus is most notable.The distemperedness of the virus-free potato production system,virus detection and virus-free seed potato supply is the main reason to the low potato yield in China.So the foremost responsibility of this research is to optimizing the virus detection system.Designing the special primers according to the conservative regions of each virus, the multiplex-RT-PCR（m-RT-PCR）strategy is adopted in this research in order to set up a single PCR system to detect six potato virus（PVX,PVY,PVA,PVM,PVS,PLRV ） and one potato virod（PSTVd） synchronously,steadily,repeatedly and exactedly.The main results are as follows:1.A single PCR system to detect six potato virus（PVX,PVY,PVA,PVM,PVS, PLRV ） and one potato virod（PSTVd） simultaneously was set up and perfected.The PCR system containsl0×PCR buffer 2μl,2.5m mol/L MgCl2 1.5μl,2mmol/L dNTP 3μl,Taq enzyme 1Unite,10μmol/L special primers of each virus（PVX and PVS 0.3μl,PVY,PVM, PLRV and PSTVd 0.5μl,PVA 0.7μl）,cDNA 2μl and the total volume is 20μl.The PCR condition is 94℃5 min,40 cycles of 94℃45 sec,57℃45 sec and 72℃90 sec,and at last 72℃10 min.2.Evaluation of developed m-RT-PCR technique was carded out by comparison with the DAS-ELISA using 20 potato in vitro plantlets,and the results showed that the m-RT-PCR system was more efficient,sensitive and economical than the DAS-ELISA.3.The virus detection Kits via m-RT-PCR technique was designed and applicable to potato virus detection. |
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