The Molecular Identification And Detection Methods Of Potato Viruses In Fujian

With the specific primers which are designed based on the Potato virus S (PVS) coat protein (CP) gene sequence (885bp) , one gene fragment (0.88kb) is amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA of the plant infected by PVS. The gene is cloned into E. call. DH5 a and sequenced. The sequence is compared with the sequence of the homologous gene of other isolates of PVS, The result shows high homology with the other isolates (the highest homology can reach 95% of nucleic acid and 98% of amino acid) and shows the virus is PVS. The CP amino acid sequences of different isolates of PVS are compared. The result shows: there is great difference on the amino acid sequence among the PVS isolates, the homology ranges from 90% to 100%. Based on CP amino acid sequence the phylogenic tree of PVS is established and the isolates are clustered into many groups.The expression vector of PVS CP gene is constructed, a 58kDa fusion protein product is expressed in E.coli DH5 a successfully. The result of western blot shows that GST-SCP can reacts with the antiserum of PVS and GST-SCP has the same immunocompetence as the natural coat protein of PVS. The antiserum of GST-SCP is produced and is used in the detection of PVS by ELISA, the sensitivity is Img fresh leaf.Four simple, quick, sensitive, reliable molecular detection techniques of potato virus S (PVS) are reported in the article: One-step RT-PCR, imminocapture-RT-PCR (IC-RT-PCR) and Nucleic acid spot hybridization (NASH) and PCR-microplate hybridization method. The one-step RT-PCR detection technique is simpler and quicker than the routine RT-PCR technique. IC-RT-PCR can detect the virus without the purification of the virus RNA. the sensitivity of the two method is as same as the routine RT-PCR's. The NASH detection method of PVS is established and is improved greatly. It is more sampler and quicker and is more suitable for the detection of a great number of samples. The PCR-microplate hybridization method can detect the virus without using EB. It has high speciality.With the specific primers which are designed based on the Potato virus X(PVX) coat protein (CP) gene sequence (71 Ibp) , one gene fragment (0.7kb) is amplified by RT-PCR using the total RNA of the plant infected by PVX. The gene is cloned into E. coli. DH5 a and sequenced. The sequence is compared with the sequence of the homologous gene of other isolates of PVX. The result shows high homology with the other isolates (the highest homology can reach 95% of nucleic acid and 97% of amino acid) and shows the virus is PVX. The CP amino acid sequences of 26 different isolates of PVX are compared. The result shows: there is great difference on the amino acid sequence among the PVX isolates, the homology ranges from 86% to 99%. Based on CP amino acid sequence, the phylogenic tree of PVX is established and the isolates are clustered into many groups.The expression vector of PVX CP gene is constructed and expressed in E. coli. DH5 a successfully. The result of SDS-PAGE shows a 52kDa fusion protein is produced. The result of western blot shows that GST-XCP can react with the antiserum of PVX and GST-XCP has the same immunocompetence as the natural coat protein of PVX. The antiserum of GST-XCP is produced and is used in the detection of PVX by ELISA, the sensitivity is Img fresh leaf.With the virus specific primers XI and X2, the routine RT-PCR ^ One-step RT-PCR and IC-RT-PCR detection methods of PVX are established. These detection methods are sensitive, quick and specific. Using the PVX CP labeled by DIG, NASH and PCR-microplate hybridization detection method of PVX are established and improved greatly. It is more sensitive and quick and suitable for the detection of a great number of samples without using EB.With the specific primers YP1 and YP2 which are designed based on the Potato virus Y (PVY) PI gene sequence, one gene fragment ( 0.83kb ) is amplified by RT-PCR using the total RNA of the plant infected by PVY. The gene is cloned into E. coli. DH5 a and se...