Establishment And Optimization Of A Multiplex PCR System For Simultaneous Detection Of 7 Potato Pathogens

China is the largest potato grower and producer in the world,but the development of this industry has been threatened by a variety of fungal,bacterial and viral pathogans.Efficient and convenient detection methods are important to protect the crop from the harm of potential pathogens,guarantee the quality of seed potato,and control the spread of these diseases.PCR technology is widely used in the detection of potato diseases with high sensitivity,especially multiple PCR technique can detect multiple target pathogens at the same time in one assay,greatly saving time and cost.Therefore,it is great helpful for potato production to develop and establish a multiple PCR system which can detect main potato pathogens quickly and accurately.This laboratory has already established a multiplex PCR system which can detect6 main potato viruses simultaneously,and multiplex PCR system for detection of both potato late blight and bacterial wilt has also been studied,but multiple PCR system for detection of both DNA（fungi,bacteria）and RNA（virus）pathogens has not been reported in potato.This study designed and identified specific PCR primers for 9 main potato pathogens,including fungal disease（late blight,black scurf,dry rot）,bacterial diseases,bacterial wilt,black shank,scab)and virus disease（PVY,and PLRV,PVX）.Through two rounds screening by simplex and multiple PCR,7 pairs primers sepecial for the potato late blight（INF1/INF2）,black scurf（BS4F/BS4R）,dry rot（Gf F/Gf R）,bacterial wilt（Rsol F/Rsol R）,PVY（3S/4A）,PLRV（4F/4R）,PVX（4F/4R）were survived,and a 7-plex PCR system was esteblished by conbining these primers in one PCR assay.Furthermor,the optimal annealing temperature,concentration of templates and primers,enzyme concentration,concentration of d NTPs and Mg²⁺,and conditions such as PCR strengthening agent were determined.The optimized multiplex PCR system established in this study has high specificity for the seven DNA and RNA pathogens,the size of amplicons of target pathogens distributed between 100 and 600 bp,the detection sensitivity of multuplex PCR assay reached 7×10^-2 ng（DNA/c DNA）templates with high repeatability.In order to further validation the applicability of the multiplex PCR,104 potato tuber samples collected in open field were tested by this system and comparing with ELISA and simplex PCR,the results showed that 6 types of pathogens have been detected,and the multiplex PCR system was more sensitive than the ELISA assay in detection of three viral pathogens,while the detection sensitivity for potato black scurf and bacterial wilt was lower than that of simplex PCR,but the multiplex PCR system can meet the demands of the production test.The new established multiplex PCR assay has realized the simultaneous detection of 7 major DNA and RNA pathogens in potato for the first time,providing us a rapid,efficient,reliable and lowcost method for detection of potato diseases,and supplied technical supports for the quality control of seed potato production and management of those potato diseases,which is helpful to promote the development of potato industry in China.