Studies On The Methods Of Detecting Virus-free Seed Potatoes By RT-PCR

Virus is an important factor that affects the potato's production, which emphsizes the need for virus-free seed potatoes. Therefore, the technology of detecting virus is very necessary. The fast methods of detecting potato virus X(PVX) and potato virus Y(PVY) by RT-PCR were studied to detect potato samples. The results were obtained as follows:(1) The RNA extraction methods were improved: chloroform was added in place of liquid nitrogen to restrain RNase when the material was grinded. The improvement was successfully applied to "single step method" and "CTAB method".(2) The total RNA was extracted by the improved "CTAB method" from potato tubers. This method could not only get rid of amylose entirely, but also operation was simple and the cost was little.(3) The nucleotide sequence of potato virus X Fujian isolate were compared with that of Beijing isolate. A pair of congenetic primers were designed in conserved region of coat protein nucleotid sequence. Therefore, the RT-PCR method of detecting PVX was builded.(4) The nucleotide sequences of potato virus Y N strain (PVYN) and O strain (PVYO) were compared. A pair of homologous primers were designedaccording to the conserved region of coat protein gene. So the RT-PCR method of detecting PVY was build whether the template was PVYN or PVYO.(5) Judging by the result of nucleotide sequences comparison between PVYN and PVYO, the 3' primer was designed in the conserved region of CP and two 5' primers were obtained in two variable places. Moreover, one of 5'primers was only according to PVYN and the other was only according to PVYO. So a complex RT-PCR method was builded which could differentiate N strain and O strain.(6) To realize simultaneously detecting PVX ,PVY, the complex RT-PCR method was primarily builded which could detect PVX,PVY at one time. (7) For reducing the experiment cost, the primary RT-PCR methods of PVX and PVY were improved. Several different disposals of reverse transcriptase (AMV) were set up when the first strand cDNA was synthesised. The result was that only 1U AMV could accomplish virus detection. The improved methods were successfully used to detect potato samples. (8) The builded methods about PVX and PVY were used to test virus-free seed potatoes "LUNYINYIHAO" and the cropped potatoes sample from "Lunan". The result manifested that these methods were sensitive and high efficient.