No abstract

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.

Aims: In this study, a loop-mediated isothermal amplification (LAMP) assay has been developed and evaluated for the rapid and sensitive detection of Verticillium dahliae Kleb., the causal agent of vascular wilts in many economically important crops. Methods and Results: LAMP primers were designed based on a previously described RAPD marker, and the LAMP assay was applied for direct detection of V. dahliae grown on medium and from soil samples without DNA purification steps (direct-LAMP). Thirty-two agricultural soil samples from various olive orchards were collected, and the presence of pathogen was detected by LAMP, direct-LAMP and nested-PCR methods. The LAMP methodology could successfully detect V. dahliae with high specificity, and cross-reaction was not observed with different pathogenic and nonpathogenic fungi and bacteria. The LAMP assay was capable of detecting a minimum of 500 and 50 fg of purified target DNA per reaction of V. dahliae ND and D pathotypes, respectively. In contrast, nested-PCR could only detect 5 pg reaction À1 for both pathotypes. In artificially infested soil samples, the LAMP method detected 5 microsclerotia per gram of soil. Conversely, nested-PCR assay detected 50 microsclerotia g À1 soil. The detection ratios of LAMP and direct-LAMP protocols were better (26 and 24 positive samples out of 32 agricultural soils analysed, respectively) than that obtained for nested-PCR method (22 positive results). Moreover, direct-LAMP yielded positive detection of V. dahliae in agricultural soil samples within 60-80 min. Conclusions:The newly developed LAMP method was proved to be an effective, simple and rapid method to detect V. dahliae without the need for either expensive equipment or DNA purification. Significance and Impact of Study: This technique can be considered as an excellent standard alternative to plating and nested-PCR assays for the early, sensitive and low-cost detection of V. dahliae as well as other soilborne pathogens in the field.

Loop-mediated isothermal amplification assay amplifies DNA/RNA with high specificity and sensitivity. In this study, we describe an optimized reverse transcription-LAMP assay for detection of Potato Leafroll Virus. Firstly, DAS-ELISA assay was performed to detect of the virus in a collection containing 40 suspicious samples. Lastly, two samples were detected as the positive samples. Then, the positive samples were verified by RT-PCR and RT-LAMP methods. Furthermore, the results demonstrated that the RT-LAMP assay was 40 times sensitive and 4 time faster compared to RT-PCR. RT-LAMP assay was accomplished in the water bath either frees from any thermal cycler machine or sophisticated laboratories facility. Moreover, in RT-LAMP reaction the positive samples were detected through turbidity which produced by magnesium pyrophosphate. Interestingly, the application of CaCl 2 instead of MgSO 4 which create calcium pyrophosphate in reaction could significantly increase both stability and concentration of turbidity. Consequently, it could be an interesting alternative to MgSO4. Overall, the newly developed RT-LAMP assay can be a sensitive, specific and low-cost method for early detection of Potato Leafroll Virus and also other viral plant pathogens.