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.

scholarly journals A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

2021 ◽  
Author(s):  
Livio M. Costa-Junior ◽  
Umer N. Chaudhry ◽  
Philip J. Skuce ◽  
Seamus Stack ◽  
Neil D. Sargison
Keyword(s):  
Genetic Susceptibility ◽  
Management Practices ◽  
Anthelmintic Resistance ◽  
Lamp Assay ◽  
Gastrointestinal Nematode ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Snp Typing ◽  
Field Samples ◽  
Β Tubulin

AbstractDevelopment of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

scholarly journals Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

2011 ◽  
Vol 47 (No. 4) ◽  
pp. 140-148 ◽  
Author(s):  
N. Rostamkhani ◽  
A. Haghnazari ◽  
M. Tohidfar ◽  
A. Moradi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Transgenic Cotton ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Leaf Tissues

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T<sub>2</sub> transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65&deg;C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10<sup>&ndash;1</sup> to 10<sup>&ndash;8</sup>) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

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.

scholarly journals Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

2020 ◽  
Vol 21 (5) ◽  
pp. 1756 ◽  
Author(s):  
Sumyya Waliullah ◽  
Kai-Shu Ling ◽  
Elizabeth J. Cieniewicz ◽  
Jonathan E. Oliver ◽  
Pingsheng Ji ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Visual Observation ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Infected Leaf ◽  
Loop Mediated Isothermal Amplification ◽  
Efficient Detection ◽  
Field Samples ◽  
Optimum Reaction ◽  
Cucurbit Leaf Crumple Virus

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus

Plant Disease ◽  
2020 ◽  
Author(s):  
Amol Kokane ◽  
Sunil Kokane ◽  
Ashish Warghane ◽  
Mrugendra G Gubyad ◽  
Ashwani Kumar Sharma ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Color Change ◽  
Lamp Assay ◽  
Single Step ◽  
Isothermal Amplification ◽  
Ringspot Virus ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the north-west of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) that is time-consuming. Here, we describe a novel, one-step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. The RT-LAMP assay was standardized by designing and testing four different primers that targeted the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. ICRSV-RT-LAMP assay developed in the present study is a simple, rapid, sensitive, and specific, technique. Moreover, the assay consists of only a single step and is more cost-effective than existing methods. This represents the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large scale indexing of field samples in diagnostic laboratories, nurseries, and for quarantine applications.

scholarly journals A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Silvia Gonçalves Mesquita ◽  
Floria Gabriela dos Santos Neves ◽  
Ronaldo Guilherme Carvalho Scholte ◽  
Omar dos Santos Carvalho ◽  
Cristina Toscano Fonseca ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Lamp Assay ◽  
Minas Gerais ◽  
Cross Reactivity ◽  
Molecular Techniques ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples ◽  
Collection Sites

Abstract Background Schistosomiasis a neglected tropical disease  endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. Methods Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. Results Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. Conclusions Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques. Graphical abstract

scholarly journals Optimization of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Schistosoma Mansoni (Trematoda: Digenea) Detection in Biomphalaria spp. from Endemic Areas for Schistosomiasis in Brazil

2021 ◽  
Author(s):  
Silvia Gonçalves Mesquita ◽  
Floria Gabriela dos Santos Neves ◽  
Ronaldo Guilherme Carvalho Scholte ◽  
Omar dos Santos Carvalho ◽  
Cristina Toscano Fonseca ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
False Negative ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Promising Alternative ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples ◽  
Endemic Areas ◽  
Collection Sites

Abstract Background: Schistosomiasis mansoni is a neglected tropical disease endemic in Brazil caused by Schistosoma mansoni, which is transmitted by Biomphalaria snails. Among all measures to control and eliminate the disease, accurate mapping and monitoring of snail breeding sites for susceptible and/or infected hosts in endemic areas are recommended. Parasitological methods are frequently used to identify infected snails, although they have many limitations, often providing false-negative results. Loop-mediated isothermal amplification (LAMP) is a promising alternative method for a more sensitive, rapid, and cost-effective diagnosis. However, standardization of LAMP assays is challenging due to the variety of parasites that are co-endemic with S. mansoni, and their varying prevalence rates in different areas. In this work, we aimed to optimize a LAMP assay for the detection of S. mansoni in Biomphalaria snails from endemic areas in the state of Minas Gerais, Brazil. Methods: A total of 1,001 snails were collected in five municipalities of the Mucuri and Jequitinhonha Valleys. Snails were pooled and squeezed according to the collection site to detect the presence of the larval forms of S. mansoni and other trematodes. Pooled snails were submitted to pepsin digestion and DNA extraction. Then molecular assays were performed for the species-specific identification and characterization of the samples. A LAMP assay was optimized and validated using laboratory and field samples. Results: Using the parasitological method, S. mansoni cercariae were detected in snails from two collection sites. Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites by PCR-RFLP. Multiplex PCR, LS-PCR, and conventional PCR allowed the detection of positive snails in four additional sites. The optimized LAMP assay was effective in detecting the presence of S. mansoni infection with 100% sensitivity, 91.66% specificity, and a Kappa index of 0.88, when compared to LS-PCR and conventional PCR. Conclusions: Our findings suggest that LAMP is a good alternative for the detection and monitoring of transmission foci of S. mansoni, as it enabled the detection of infection three times more than the parasitological examination and is more applicable directly in the field when compared to other molecular approaches.

RAPID DIAGNOSIS OF BLUETONGUE VIRUS SEROTYPES 2 AND 12 INFECTION BY REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION

2015 ◽  
Vol 41 (03) ◽  
pp. 187-196
Author(s):  
Jia-Ling Yang ◽  
Yi-Chia Li ◽  
Shu-Chia Hu ◽  
Fan Lee ◽  
Fun-In Wang
Keyword(s):  
Reverse Transcription ◽  
Bluetongue Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Universal Primers ◽  
Subclinical Infection ◽  
Loop Mediated Isothermal Amplification ◽  
Mouth Disease Virus ◽  
Field Samples

Bluetongue (BT), an arthropod-borne viral disease, is caused by the bluetongue virus (BTV), belonging to the genus Orbivirus of the family Reoviridae. Most species of ruminants are susceptible to BTV, but most infections go subclinical. These 'reservoir hosts' may potentially further increase the viral transmission and expansion of the disease; thus, detection of subclinical infection is important. To detect the BTV, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed using primers targeted to six regions of the segment 5 (NS1) gene of the BTV2/KM/2003. The assay was completed in 1 h at a temperature 65°C, and the products were specifically digested with MboII enzyme presented in the target region. The in vitro sensitivity of the RT-LAMP was 100 copies, characterized by a qRT-PCR. The RT-LAMP did not cross-react with four tested common ruminant infectious agents, namely foot and mouth disease virus, goat pox virus, bovine herpesvirus 1, and Clostridium perfrigens. The RT-LAMP was applied to whole blood samples from 15 clinically healthy dairy cattle, and was able to detect BTV from 3/15 animals, and in particular 1 of the 3 animals was seronegative by cELISA. Positive RT-LAMP samples were reproducible. This RT-LAMP provides a simple, efficient, and sensitive method to specifically detect BTV and is suitable for the screening of field samples with a potential to pick up subclinical infection. The alignments of the outer primer region indicated matches of > 85% with 18 out of 26 BTV serotypes, implying its potential as universal primers.

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.

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