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For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: This study reports the development of a loop-mediated isothermal amplification (LAMP) reaction for the detection of Pythium myriotylum. The primer set targeting the ITS sequence of P. myriotylum worked most efficiently at 60°C and allowed the detection of P. myriotylum DNA within 30 min by fluorescence monitoring using a real-time PCR instrument. The peak denaturing temperature of amplified DNA was about 87·0°C. In specificity tests using eight Pythium myriotylum strains, 59 strains from 39 species of Pythium, 11 Phytophthora strains and eight other soil-borne pathogens, LAMP gave no cross-reactions. The detection limit was 100 fg of genomic DNA, which was as sensitive as PCR. LAMP could detect P. myriotylum in hydroponic solution samples, and the results coincided with those of the conventional plating method in almost all cases. The LAMP method established in this study is a simple and sensitive tool for the detection of P. myriotylum., Significance and Impact of the Study: This study shows the first LAMP assay for the detection of Pythium myriotylum. The primer set designed from ITS region of P. myriotylum can detect the pathogen in field sample with a fast and convenient method. Analysis of the annealing curve of the LAMP reaction products increases the reliability of the LAMP diagnosis. This study shows that the diagnostic method using the LAMP assay is useful for monitoring P. myriotylum in the field., (© 2014 The Society for Applied Microbiology.)

Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides, and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection of P. helicoides., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65 °C and could detect each virus RNA within 10 min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6 °C, 84.8 °C and 86.4 °C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of peach latent mosaic viroid (PLMVd) was developed. Four primer sets (OLD, OLD1, NEW, and Fukuta's) were designed originally. Based on initial experiments the set OLD1 was selected for further evaluation. Simple and accelerated RT-LAMP was preformed using degenerate and no degenerate forward-loop (F-loop) and backward-loop (B-Loop) primers. Degenerate primers were selected, and after determination of their best concentration (0.8microM), the reaction was preformed at different temperatures (60-67.5 degrees C) using three different betaine concentrations (0.8M, 0.4M, and 0.2M). Optimal conditions were found to be 62.5 degrees C and 0.8M betaine. Under these conditions, using tRNA as template, PLMVd was detected within only 32min, compared to 180min of RT-PCR, using the Real Time Turbimeter (LA200, Teramecs) which measures the turbidity caused by the production of insoluble magnesium pyrophosphate. In addition, RT-LAMP was more sensitive than RT-PCR. PLMVd was detected in peach, plum, apricot, pear, wild pear and quince samples.

In this study, LAMP markers linked to shelf-life in melon (Cucumis melo L.) were developed by converting a cleaved amplified polymorphic sequences (CAPS) marker (C2). The CAPS-PCR fragments from the long-shelf-life melon (O-3) and short-shelf-life melon (Nat-2) were cloned and sequenced to construct LAMP primers. A single nucleotide polymorphism (SNP) was identified between O-3 and Nat-2. LAMP primers were designed to detect the SNP. In the LAMP reaction to detect long-shelf-life melon, the turbidity of the templates using O-3, F1, homozygous long-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. In contrast, the turbidity of Nat-2 and homozygous short-shelf-life F2 lines did not increase even after 90 min. In the LAMP reaction to detect short-shelf-life melon, the turbidity of the templates using Nat-2, F1, homozygous short-shelf-life F2 lines and heterozygous long-shelf-life F2 lines started to increase after 40 min. But the turbidity of O-3 and homozygous long-shelf-life F2 lines did not increase after 90 min. This attests to the high reliability and usefulness of LAMP for marker-assisted selection.

An immunocapture reverse transcription loop-mediated isothermal amplification (IC/RT-LAMP) was developed for the detection of tomato spotted wilt virus (TSWV) from chrysanthemum. This method enabled sensitive, reproducible and specific detection of TSWV from chrysanthemum plants. In the RT-LAMP method, TSWV genomic RNA could be amplified under isothermal (65 degrees C) conditions within 1 h. The resulting amplicons were detected by the measurement or observation of the turbidity of the reaction mixture without gel electrophoresis. IC/RT-LAMP was 100 times more sensitive than IC/RT-PCR.

The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of amplification of TYLCV genomic DNA without gel electrophoresis.

Arapid and simple procedure is described to detect the genomic RNA molecule of Japanese yam mosaic potyvirus (JYMV). This method, named RT-LAMP, allows direct detection of RNA from infected plants without careful RNA extraction, rapid thermal cycling and gel electrophoresis. RT-LAMP was successfully applied to leaves, propagules and roots of Japanese yam infected with JYMV. One of the characteristics of the RT-LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophospate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of the amplification of JYMV genomic RNA without gel electrophoresis.