scholarly journals Novel Multiplex and Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Species and Mating-Type Identification of Oculimacula acuformis and O. yallundae (Causal Agents of Cereal Eyespot), and Application for Detection of Ascospore Dispersal and in planta Use

2020 ◽  
Author(s):  
Kevin M. King ◽  
Gavin J. Eyres ◽  
Jon West ◽  
Clara Siraf ◽  
Pavel Matusinsky ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Mating Type ◽  
Field Experiments ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
In Planta ◽  
Loop Mediated Isothermal Amplification ◽  
Multiplex Pcr Assay ◽  
Species Specific ◽  
Oculimacula Acuformis

Eyespot, caused by the related fungal pathogens Oculimacula acuformis (OA) and O. yallundae (OY), is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between OA and OY (e.g. host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of OA and OY isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed allowing simultaneous determination of both species and mating type. This multiplex-PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting beta-tubulin sequences is described, which allow fast (<9 min) species-specific discrimination of global OA and OY isolates. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type specific LAMP assays were also developed for rapid (<12 min) genotyping of OA and OY isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.

Loop-mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens

Plant Disease ◽  
2021 ◽  
Author(s):  
Xiayan Pan ◽  
Xiao Wang ◽  
Junjie Yu ◽  
Mina Yu ◽  
Huijuan Cao ◽  
...  
Keyword(s):  
Mating Type ◽  
Genomic Dna ◽  
High Efficiency ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Villosiclava Virens ◽  
False Smut ◽  
Mating Type Genes ◽  
Polymerase Chain

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicle of rice. V. virens is a heterothallic ascomycete that controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect mating type of V. virens easily and rapidly by using specific primers designed on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay required only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1, and 200.0 pg of MAT1-2), which was 10-fold more sensitive than polymerase chain reaction (PCR). In addition, the application of mating type using LAMP assay was demonstrated feasibly by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggested it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

scholarly journals Detection of the Benthic Dinoflagellates, Ostreopsis cf. ovata and Amphidinium massartii (Dinophyceae), Using Loop-Mediated Isothermal Amplification

2021 ◽  
Vol 9 (8) ◽  
pp. 885
Author(s):  
Eun Sun Lee ◽  
Jinik Hwang ◽  
Jun-Ho Hyung ◽  
Jaeyeon Park
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Benthic Dinoflagellates ◽  
Field Samples ◽  
Its Gene ◽  
Buffer Composition ◽  
Species Specific

For the in situ and sensitive detection of benthic dinoflagellates, we have established an integrated loop-mediated isothermal amplification (LAMP) assay based on Ostreopsis cf. ovata and Amphidinium massartii. To detect the two species, a set of species-specific primers was constructed between the ITS gene and D1–D6 LSU gene, and the reaction temperature, time, and buffer composition were optimized to establish this method. In addition, the specificity of the LAMP primers was verified both in strains established in the laboratory and in field samples collected from the Jeju coastal waters, Korea. With the LAMP assay, the analysing time was within 45 to 60 min, which may be shorter than that with the conventional PCR. The detection sensitivity of the LAMP assay for O. cf. ovata or A. massartii was comparable to other molecular assays (PCR and quantitative PCR (qPCR)) and microscopy examination. The detection limit of LAMP was 0.1 cell of O. cf. ovata and 1 cell of A. massartii. The optimized LAMP assay was successfully applied to detect O. cf. ovata and A. massartii in field samples. Thus, this study provides an effective method for detecting target benthic dinoflagellate species, and could be further implemented to monitor phytoplankton in field surveys as an altenative.

Species-specific detection of medically important aspergilli by a loop-mediated isothermal amplification method in chronic pulmonary aspergillosis

2019 ◽  
Vol 57 (6) ◽  
pp. 703-709
Author(s):  
Kazuya Tone ◽  
Junko Suzuki ◽  
Mohamed Mahdi Alshahni ◽  
Kazuyoshi Kuwano ◽  
Koichi Makimura
Keyword(s):  
Sensitivity And Specificity ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Detection ◽  
Pulmonary Aspergillosis ◽  
Loop Mediated Isothermal Amplification ◽  
Group A ◽  
Chronic Pulmonary Aspergillosis ◽  
Species Specific

AbstractChronic pulmonary aspergillosis (CPA) is a common subtype of pulmonary aspergillosis and a life-threatening disease. However, its diagnosis remains difficult due to the lack of specific clinical features and radiologic findings, as well as the difficulty of isolating Aspergillus spp. We developed a novel species-specific detection method of medically important aspergilli using a loop-mediated isothermal amplification (LAMP) for CPA. Specific LAMP primer sets for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans were designed. The use of the LAMP assay was validated using respiratory specimens (CPA cases, n = 21; nonaspergillosis cases, n = 23). A total of 15 cases were positive in the CPA group (A. fumigatus, n = 5; A. flavus, n = 1; A. niger, n = 1; A. terreus, n = 7; A. nidulans, n = 1), but only three in the non-CPA group (A. niger, n = 2; A. terreus n = 1). The sensitivity and specificity of the diagnosis of CPA by the LAMP system were 71.4% and 87.0%, respectively. In conclusion, we developed a species-specific detection approach for five medically important aspergilli using the LAMP method. The system showed high sensitivity and specificity for diagnosis of CPA.

Diagnosis of osteoarticular tuberculosis: multi-targeted loop-mediated isothermal amplification assay versus multiplex-PCR

2021 ◽  
Author(s):  
Anish Khan ◽  
Ekta Kamra ◽  
Raj Singh ◽  
Vikrant Sharma ◽  
Vishwajeet Singh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multiplex Pcr ◽  
Lamp Assay ◽  
Cost Effective ◽  
Test Methods ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Osteoarticular Tuberculosis ◽  
Cost Effective Method ◽  
Amplification Assay

Aim: Diagnosis of osteoarticular tuberculosis (OATB) is quite challenging and there is an urgent need to design a prompt and precise diagnostic test. Methods: We developed a multi-targeted loop-mediated isothermal amplification (LAMP) assay using mpt64 (Rv1980c) and pstS1 (Rv0934) targets for the detection of Mycobacterium tuberculosis in OATB patients. Results: The sensitivities of 100 and 82.4% were obtained in confirmed (n = 10) and suspected (n = 57) OATB cases, respectively by multi-targeted LAMP with a specificity of 96.9% (n = 33). Moreover, the sensitivities attained by multi-targeted LAMP in total OATB cases were significantly higher (p < 0.05–0.01) than multiplex-PCR ( mpt64 +  pstS1) and GeneXpert assay. Conclusion: Our LAMP is simple, reliable and cost-effective method, which may develop into an attractive diagnostic kit for early detection of OATB cases.

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