Recombinase polymerase amplification assay for rapid on-site diagnosis of pear fire blight pathogen Erwinia amylovora
Erwinia amylovora is an imported quarantine plant pathogen in China,which infects Rosaceae plants such as pear,apple,hawthorn,causing pear fire blight disease.This disease,firstly discovered in the United States in 1780,has spread to many countries in the world and posed a threat to domestic pomefruits production.Strict quarantine is one of the important methods to control pear fire blight in the world.Chinese plant quarantine departments basically conducted Koch's postulate,molecular detection and rapid detection on-site following the typical symptoms to detect the pathogen.In this study,a recombinase polymerase amplification with real-time fluorescent(real-time RPA)and with lateral flow dipstick(RPA-LFD)detection methods for E.amylovora were established,which provided technical support for rapid on-site diagnosis both in field and laboratory.Based on E.amylovora 16S-23S ITS region,we designed specific primers and probes and established real-time RPA and RPA-LFD detection methods.At 39℃,both methods showed good specificity and can give results in 20 mins and 10 mins,respectively.Real-time RPA detection limit of E.amylovora strain gDNA can reach 1.38 × 10-2 ng/μL,while RPA-LFD detection limits of E.amylovora suspension and its gDNA can reach 104 cfu/mL and 1.38 × 10-3 ng/μL,respectively.RPA-LFD is accurate and can easily perform the test at the point of need.
pear fire blightrapid on-side diagnosisrecombinase polymerase amplification(RPA)real-time RPARPA-LFD
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