Rapid Detection Of Three Main Phytophthora Spp.using Lateral Flow Dipstick-recombinase Polymerase Amplification Assay

Phytophthora blight,caused by Phytophthora spp.,is one of the most devastating diseases in the world.Phytophthora spp.has the characteristics of rapid spread,outbreaks,and difficulties in prevention and control,and the direct economic losses are as high as tens of billions in crops.Establishing a rapid and accurate detection method for Phytophthora spp.is important for the early diagnosis and timely prevention and control of the disease,and is also the most important issue for ensuring the quality and yield of crops.1.Compared with the LAMP,one of a nucleic acid isothermal amplification technology,the recombinase polymerase amplification(RPA)technology combine with lateral flow dipstick(LFD)formed the LFD-RPA system,which can directly judge the detection results by eyes.This study took Phytophthora infestans,Phytophthora colocasiae,and Peronophythora litchii,three species of Phytophthora as test objects,aiming to establish the LFD-RPA rapid detection system and to optimize.The main research contents and results are as follows:2.Establishment and evaluation of Phytophthora infestans detection based on LFD-RPA technology.In this assay,preliminary designed and screened primers for the specific sequence of Phytophthora infestans,andobtained the primers(Pi LFD-RPA-F/R)for the sequence of GTP-blinding protein gene(Ypt1)and probe(Pi LFD-RPA-P)to establish a LFD-RPA detection system for Phytophthora infestans.The amplification time and temperature of the system were further optimized,and the results showed that the shortest time for the detection of test band with dark and clear band was 15 min,and the lowest temperature was 34°C.In the system sensitivity test,the lowest detectable DNA concentration for 50?l reaction volume was1pg/?l.3.Establishment and evaluation of Phytophthora colocasiae detection based on LFD-RPA technology.In this assay,preliminary designed and screened primers for the specific sequence of Phytophthora colocasiae,and obtained the primers(Pco LFD-RPA-F/R)for the sequence of GTP-blinding protein gene(Ypt1)and probe(Pco LFD-RPA-F/R)to establish a LFD-RPA detection system for Phytophthora colocasiae.The amplification time and temperature of the system were further optimized.The results showed that the shortest time for the detection of test band with dark and clear band was20 min,and the lowest temperature was 34°C.In the system sensitivity test,the lowest detectable DNA concentration for 50?l reaction volume was1pg/?l.4.Establishment and evaluation of Peronophythora litchii detection based on LFD-RPA technology.In this assay,preliminary designed and screened primers for the specific sequence of Peronophythora litchii andobtained the primers(Pl LFD-RPA-F/R)for the sequence of Rh-ammonium transporter and probe(Pl RPA-F/R)to establish a LFD-RPA detection system for Peronophythora litchii.The amplification time and temperature of the system were further optimized.The results showed that the shortest time for the detection of test band with dark and clear band was 20 min,and the lowest temperature was 34°C.In the system sensitivity test,the lowest detectable DNA concentration for 50?l reaction volume was 10pg/?l.In summary,this study provides a new detection method for the rapid identification and early diagnosis of P.infestans,P.colocasiae and Peronophythora litchii,which has strong specificity,high sensitivity,short reaction time.The temperature of the human body can be used for amplification,and the results are visible to the eye and have strong practicality.It provides an effective means for rapid detection and identification of pathogens in the field.