| Potato virus disease is an important disease affecting the potato industry,and virus detection is the basis of prevention and control.In this study,we established Reverse transcription-recombinase polymerase amplification（RT-RPA）detection assay for potato leaf roll virus（PLRV）and potato virus S（PVS）,established a triple RT-RPA detection assay for potato virus Y（PVY）,PLRV and PVS,and try to achieve the field detection for PVY.The results of this study are mainly as follows:1.The RT-RPA detection assay for PLRV and PVS was established while four pairs of RPA primers were designed and screened respectively.And the assay allowed specific detection for PLRV and PVS.When the plasmids were used as templates,the sensitivity of PLRV detection was 1 fg·μL-1and the sensitivity of PVS detection was 100 ag·μL-1,which were both 10 times higher than PCR.The incubation time and temperature were optimized and showed that RT-RPA for PLRV detection and PVS detection should be incubated over 10 min at 25℃or 5 min from 30 to 40℃.The results of RT-RPA detection of 20 field samples of PLRV and PVS are completely consistent with ELISA and RT-PCR.2.The triple RT-RPA assay was established to diagnose the separate or compound infection of PVY,PLRV and PVS.The primer concentrations as PVY 800 n M,PLRV 240n M and PVS 160 n M allowed to balance detection of PVY,PLRV and PVS.When the plasmids was used as templates,the sensitivity of the triple RT-RPA assay was 1 fg·μL-1,which is equal to PCR.The incubation time and temperature were optimized and showed that the triple RT-RPA assay should be incubated over 30 min at 25℃,10 min at 30℃,or5 min from 35 to 40℃to detect PVY,PLRV and PVS both.The results of the triple RT-RPA detection of 20 field samples are completely consistent with the triple RT-PCR,while ELISA have 3 inconsistent results.3.The exploration of the field detection for PVY includes three contents.First,the direct-antigen capture method was optimized to complete the preparation of the nucleic acid amplification template in 1 min from 0 to 35℃.And the sensitivity by RT-PCR detection using PVY RNA extraction by direct-antigen capture method is equal to ELISA detection.Secondly,the one-step RT-RPA detection assay was established,witch detection sensitivity is equal to two-step RT-RPA and two-step RT-PCR.The incubation time and temperature were optimized and showed that the one-step RT-RPA for PVY detection should be incubated over 20 min at 30℃or 10 min from 35 to 40℃.Finally,the lateral flow dipsticks,nucleic acid dyes,metal indicator and acid-base indicator visualize the RPA products were tried,only to not effectively visualize the RPA products. |
