No abstract

A reliable and rapid pathogen detection protocol that utilizes loop-mediated isothermal amplification (LAMP) was developed for detection of Erwinia amylovora, the casual agent of fire blight. The six LAMP primers applied were derived from the highly conserved fragment of the chromosomally amsH gene. Despite the proposed LAMP as well as nested PCR presenting equal values of sensitivity (2×10 1 CFU/ml or more) for pure cultures, as compared with conventional PCR (2×10 3 CFU/ml), both methods were together superior. The specificity assay also showed that the LAMP protocol is species-specific for detection of E. amylovora even in inter-species analysis. Meanwhile, when all 208 naturally infected samples were examined, the specificity value of LAMP was 84%, while conventional and nested PCR could detect only 59% and 73% of the whole collection. Significantly, an independent behaviour versus host plant as well as each strain origin was also observed regarding the current LAMP method as well as other two PCR-based methods. All the results, overall, indicated that the LAMP offers an interesting novel and convenient assay format for the quick and specific chromosomal detection and diagnostic tool of recognition of E. amylovora and therefore presents an alternative to PCR-based assays.

Loop‐mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) to visually detect Potato leafroll virus. One‐step RT‐LAMP was performed using RNA of PLRV‐infected potato leaves and a set of primers (F3, B3, FIP, BIP, LF and LB) designed for RT‐LAMP reaction of the coat protein (CP) gene of PLRV. Positive effects of RT‐LAMP were detected by agarose gel electrophoresis and hydroxynaphthol blue (HNB) dye and were shown by a colour change from violet to sky blue. RT‐LAMP with HNB dye proved to be a simple assay for the rapid detection of PLRV.

To diminish the time required for some diagnostic assays including reverse transcription PCR (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP; due to mainly RNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture reverse transcription (IC/RT-PCR) protocol on the basis of Potato Leafroll virus (PLRV) genome were used and optimized. In this regard, all six IC-RT-LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB) together with IC/RT-PCR primers were designed on the basis of the highly conserved sequence (ORF3) of coat protein gene (GenBank accession number: U73777) of PLRV genome. Even though DAS-ELISA, IC/RT-PCR and IC-RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect IC-RT-LAMP products, both hydroxynaphthol blue and GeneFinder™ could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. Altogether, as IC-RT-LAMP is sensitive, cost-effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PLRV recognition and probably other viral-based diseases.