Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria solani

Abstract

Early blight (EB), caused by the pathogen Alternaria solani, is a major threat to global potato and tomato production. Early and accurate diagnosis of this disease is therefore important. In this study, we conducted a loop-mediated isothermal amplification (LAMP) assay, as well as conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (RT-qPCR) assays to determine which of these techniques was less time consuming, more sensitive, and more accurate. We based our assays on sequence-characterized amplified regions of the histidine kinase gene with an accession number (FJ424058). The LAMP assay provided more rapid and accurate results, amplifying the target pathogen in less than 60 min at 63°C, with 10-fold greater sensitivity than conventional PCR. Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR. qPCR was the most sensitive among the assays evaluated, being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration (100 fg). The LAMP assay was more sensitive than conventional PCR, but less sensitive than nested PCR and qPCR; however, it was simpler and faster than the other assays evaluated. Despite of the sensitivity, LAMP assay provided higher specificity than qPCR. The LAMP assay amplified A. solani artificially, allowing us to detect naturally infect young potato leaves, which produced early symptoms of EB. The LAMP assay also achieved positive amplification using diluted pure A. solani culture instead of genomic DNA. Hence, this technique has greater potential for developing quick and sensitive visual detection methods than do other conventional PCR strategies for detecting A. solani in infected plants and culture, permitting early prediction of disease and reducing the risk of epidemics.

Comparative evaluation of lateral flow immunoassays, LAMP, and quantitative PCR for diagnosis of fire blight in apple orchards

“…5). The sensitivity of LAMP and qPCR were identified to be higher than conventional d i a g n o s t i c m e t h o d s f o r A l t e r n a r i a s o l a n i a n d Phytophthora infestans detection from potato, tomato, and other related host plants (Okiro et al 2019;Khan et al 2018;Lees et al 2019). The real-time PCR has previously also been identified as the most sensitive method, followed by LAMP assay, to detect the Xylella fastidiosa pathogen from blueberry (Waliullah et al 2019).…”

Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics

“…Second, isothermal amplification is more sensitive and faster than PCR [28] as it does not rely on discrete thermal cycles like PCR, but rather involves continuous amplification that can result in detectable amplicons within 10 minutes. Furthermore, there are several reports that suggest that isothermal technologies such as LAMP with 6-8 primer binding sites can result in greater specificity than PCRbased methods [29,30]. The combination of isothermal amplification with recent advances in diagnostic technologies such as the equipment-free dipstick nucleic acid purification technology [31] and naked eye-based readout technology [32], will facilitate the development of PONcapable molecular diagnostic platforms.…”

Isolation and molecular identification of Rhizoctonia solani and Fusarium solani isolated from cucumber (Cucumis sativus L.) and their control feasibility by Pseudomonas fluorescens and Bacillus subtilis

“…Polymerase chain reaction (PCR) technology was used in this study to diagnose the isolates of F. solani and R. solani due to its high accuracy in the diagnosis of many organisms, including pathogenic and non-pathogenic fungi such as F. solani, R. solani, Alternaria alternata, and Aspergillus spp. (AL-Abedy et al 2018;Khan et al 2018).…”