scholarly journals Development of a Loop-Mediated Isothermal Amplification Assay as an Early-Warning Tool for Detecting Emerald Ash Borer (Coleoptera: Buprestidae) Incursions

2020 ◽  
Vol 113 (5) ◽  
pp. 2480-2494 ◽  
Author(s):  
George Kyei-Poku ◽  
Debbie Gauthier ◽  
Guoxing Quan
Keyword(s):  
Large Scale ◽  
Insect Pest ◽  
Emerald Ash Borer ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Agrilus Planipennis ◽  
Isothermal Amplification ◽  
Insect Species ◽  
Coi Gene ◽  
Loop Mediated Isothermal Amplification

Abstract The emerald ash borer (EAB), Agrilus planipennis (Fairmaire), is the most destructive invasive insect species of ash (Fraxinus spp.) in North America. An accurate method for early detection of this noxious insect pest is indispensable to providing adequate warning of A. planipennis infestation. A loop-mediated isothermal amplification (LAMP) assay (EAB-LAMP) was developed based on mitochondrial cytochrome c oxidase subunit I (COI) gene. The EAB-LAMP required only 30 min at 65°C to amplify A. planipennis DNA from specimens collected from geographically distinct locations. There was no cross-reactivity with other Agrilus and insect species. The developed EAB-LAMP differentially detected traces of A. planipennis genome (COI) within frass from various Fraxinus species. EAB-LAMP was also able to distinguish among A. planipennis DNA and other Agrilus species and nontarget insect species in trap captures. By detecting A. planipennis DNA in two additional trap captures (in situ), the EAB-LAMP was more sensitive and reliable than visual inspection. We tested the quantitative nature of the assay by evaluating pooled trap samples and demonstrated that the EAB-LAMP was capable of functioning optimally using a pool size of at least five individual trap samples. This potentially circumvents the need to perform large-scale individual analysis for processing trap samples. Considering its performance, specificity, sensitivity, and repeatability, the developed EAB-LAMP could be a valuable tool to support strategy and operation of large-scale surveillance for A. planipennis and could profitably be used in routine monitoring programs for effective management of A. planipennis.

scholarly journals Rapid Visualization and Detection of Staphylococcus Aureus Based on Loop-Mediated Isothermal Amplification

2021 ◽  
Author(s):  
Chuan Wu ◽  
Yuanyuan Zeng ◽  
Yang He
Keyword(s):  
Staphylococcus Aureus ◽  
Limit Of Detection ◽  
Reaction System ◽  
Bacterial Pathogen ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Diagnostic Efficacy ◽  
Diverse Range ◽  
Loop Mediated Isothermal Amplification

Abstract Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 16 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by 10-fold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP. The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections.

scholarly journals Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study

2020 ◽  
Author(s):  
Azeem Mehmood Butt ◽  
Shafiqa Siddique ◽  
Xiaoping An ◽  
Yigang Tong
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification ◽  
Qualitative Detection ◽  
Detection Assay ◽  
Testing Protocols ◽  
Positive Results

AbstractSevere acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) and 25 non-COVID-19 cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive results in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.

scholarly journals Pneumocystis jirovecii Colonization and Its Association with Pulmonary Diseases Based on a Multicenter Study by an Improved Loop-Mediated Isothermal Amplification Assay

2019 ◽  
Author(s):  
Ting Xue ◽  
Zhuang Ma ◽  
Fan Liu ◽  
Wei-Qin Du ◽  
Li He ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Cross Reactivity ◽  
Interstitial Lung Diseases ◽  
Isothermal Amplification ◽  
Pneumocystis Jirovecii ◽  
Pulmonary Diseases ◽  
Chronic Obstructive ◽  
Hiv Patients ◽  
Loop Mediated Isothermal Amplification ◽  
Cell Counts

Abstract Background Pneumocystis jirovecii ( P. jirovecii ) is an opportunistic fungal pathogen and the role of its colonization in pulmonary diseases has become a popular focus in recent years. The aim of this study is to develop an improved loop-mediated isothermal amplification (LAMP) assay for detection of Pneumocystis jirovecii ( P. jirovecii ) DNA and use it to examine the prevalence and association of P. jirovecii colonization among non-HIV patients with various pulmonary diseases. Methods We modified the previously reported LAMP assay for P. jirovecii by adding real-time detection. This method was used to detect P. jirovecii colonization in pulmonary samples collected from 403 non-HIV patients with various pulmonary diseases enrolled from 5 hospitals in China. We determined the prevalence of P. jirovecii colonization in 7 types of pulmonary diseases and assessed the association of P. jirovecii colonization with clinical characteristics of these diseases. Results The new LAMP assay showed no cross-reactivity with other common pulmonary microbes and was 1,000 times more sensitive than that of conventional PCR. Using the new LAMP assay, we detected P. jirovecii colonization in 281 (69.7%) of the 403 patients enrolled. P. jirovecii colonization was more common in interstitial lung diseases than in chronic obstructive pulmonary disease (COPD) (84.6% vs 64.5%, P < 0.05). Patients with acute exacerbation of COPD had a higher prevalence of P. jirovecii colonization compared to patients with stabilized COPD (67.4% vs 43.3%, P < 0.05). P. jirovecii colonization was associated with decreased pulmonary function, increased levels of 1,3-β-D-glucan and C-reactive protein, and decreased levels of CD4+ T-cell counts (P < 0.05 for each). Approximately 70% of P. jirovecii colonized patients had confections with other fungi or bacteria. Conclusions We developed an improved LAMP assay for detecting P. jirovecii . Our multi-center study of 403 patients supports that P. jirovecii colonization is a risk factor for the development of pulmonary diseases and highlights the need to further study the pathogenesis and transmission of P. jirovecii colonization in pulmonary diseases.

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 Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2

2020 ◽  
Author(s):  
Gun-Soo Park ◽  
Keunbon Ku ◽  
Seung-Hwa Baek ◽  
Seong Jun Kim ◽  
Seung Il Kim ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Detection Method ◽  
Colorimetric Detection ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Genomic Rna ◽  
Loop Mediated Isothermal Amplification ◽  
Human Coronaviruses ◽  
High Level

AbstractEpidemics of Coronavirus Disease 2019 (COVID-19) now have more than 100,000 confirmed cases worldwide. Diagnosis of COVID-19 is currently performed by RT-qPCR methods, but the capacity of RT-qPCR methods is limited by its requirement of high-level facilities and instruments. Here, we developed and evaluated RT-LAMP assays to detect genomic RNA of SARS-CoV-2, the causative virus of COVID-19. RT-LAMP assays in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human Coronaviruses was not observed. We also adapted a colorimetric detection method for our RT-LAMP assay so that the tests potentially performed in higher throughput.

scholarly journals Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Diagnosis of Schistosoma mansoni Infection in Faecal Samples

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Ibrahim N. Mwangi ◽  
Eric L. Agola ◽  
Robert M. Mugambi ◽  
Esther A. Shiraho ◽  
Gerald M. Mkoji
Keyword(s):  
Schistosoma Mansoni ◽  
Color Change ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Repeat Sequence ◽  
Kappa Coefficient ◽  
Isothermal Amplification ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Faecal Samples

Human intestinal schistosomiasis is caused by the blood fluke, Schistosoma mansoni. With intensified efforts to control schistosomiasis by mass drug administration using praziquantel (PZQ), there is an urgent need to have accessible, quality-assured diagnostic tests for case detection and disease surveillance and for monitoring efficacy of treatment and other interventions. Current diagnostic tools are limited by suboptimal sensitivity, slow turn-around-time, affordability, and inability to distinguish current from past infections. We describe a simple and rapid diagnostic assay, based on the loop-mediated isothermal amplification (LAMP) technology for diagnosis of S. mansoni infection in human faecal samples. The LAMP primers used in this assay were previously described and they target a 121-bp DNA repeat sequence in S. mansoni. The LAMP assay was optimized at an isothermal temperature of 63°C for 1 hour. The amplified DNA was either visualized under ultraviolet light after electrophoresis or by directly observing the color change after staining the amplicons with CYBR Green dye. The LAMP assay was evaluated against the microscopy-based procedure and the results were analysed using Cohen’s kappa coefficient to determine the degree of agreement between the two techniques. The LAMP assay reliably detected S. mansoni ova DNA in faecal samples and parasite DNA in amounts as low as 32fg. When the assay was tested for specificity against other faecal-based soil-transmitted helminths (STH), no cross-reactivity was observed. The LAMP assay was superior to the Kato-Katz assay with a 97% specificity; a high positivity score reliably detecting S. mansoni and a Kappa Coefficient of 0.9 suggested an exceptional agreement between the two techniques. The LAMP assay developed has great potential for application in field settings to support S. mansoni control and elimination campaigns.

scholarly journals Loop-Mediated Isothermal Amplification Assay for Detection of Generic and Verocytotoxin-ProducingEscherichia coliamong Indigenous Individuals in Malaysia

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Cindy Shuan Ju Teh ◽  
Kek Heng Chua ◽  
Yvonne Ai Lian Lim ◽  
Soo Ching Lee ◽  
Kwai Lin Thong
Keyword(s):  
Large Scale ◽  
Lamp Assay ◽  
Asymptomatic Infection ◽  
Isothermal Amplification ◽  
Indigenous Populations ◽  
Bacterial Strains ◽  
E Coli ◽  
Loop Mediated Isothermal Amplification ◽  
Threshold Time ◽  
Amplification Assay

We have successfully developed a Loop-mediated isothermal amplification(LAMP) assay that could specifically detect genericEscherichia coli(E. coli). This assay was tested on 85 bacterial strains and successfully identified 54E. colistrains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt = 31.12). We have also detected 46 genericE. colifrom 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producingE. coli(VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.

scholarly journals Pneumocystis jirovecii Colonization and Its Association with Pulmonary Diseases Based on a Multicenter Study by an Improved Loop-Mediated Isothermal Amplification Assay

2019 ◽  
Author(s):  
Ting Xue ◽  
Zhuang Ma ◽  
Fan Liu ◽  
Wei-Qin Du ◽  
Li He ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Pneumocystis Jirovecii ◽  
Pulmonary Diseases ◽  
Chronic Obstructive ◽  
Hiv Patients ◽  
Loop Mediated Isothermal Amplification ◽  
Cell Counts

Abstract Abstract Background Pneumocystis jirovecii is an opportunistic pathogen fungus and the role of its colonization in respiratory diseases has become a popular focus in recent years. The aim of present study is to develop an improved loop-mediated isothermal amplification (LAMP) assay for detection of Pneumocystis jirovecii (P. jirovecii) DNA and use it to examine the prevalence and association of P. jirovecii colonization among non-HIV patients with various pulmonary diseases. Methods We modified the previously reported LAMP assay for P. jirovecii by adding real-time detection. This method was used to detect P. jirovecii colonization in respiratory samples collected from 403 non-HIV patients with various respiratory diseases enrolled from 5 hospitals in China. We determined the prevalence of P. jirovecii colonization in 7 types of pulmonary diseases and assessed the association of P. jirovecii colonization with clinical characteristics of these diseases. Results The new LAMP assay showed no cross-reactivity with other common respiratory microbes and was 1,000 times more sensitive than that of conventional PCR. Using the new LAMP assay, we detected P. jirovecii colonization in 281 (69.7%) of the 403 patients enrolled. P. jirovecii colonization was more common in interstitial lung diseases than in chronic obstructive pulmonary disease (COPD) (84.6% vs 64.5%, P < 0.05). Patients with acute exacerbation of COPD had a higher prevalence of P. jirovecii colonization compared to patients with stabilized COPD (67.4% vs 43.3%, P < 0.05). P. jirovecii colonization was associated with decreased pulmonary function, increased levels of 1,3-β-D-glucan and C-reactive protein, and decreased levels of CD4+ T-cell counts (P < 0.05 for each). Approximately 70% of P. jirovecii colonized patients had confections with other fungi or bacteria. Conclusions We developed an improved LAMP assay for detecting P. jirovecii. Our multi-center study of 403 patients supports that P. jirovecii colonization is a risk factor for the development of pulmonary diseases and highlights the need to further study the pathogenesis and transmission of P. jirovecii colonization in pulmonary diseases. Keywords: P. jirovecii; colonization; respiratory diseases; loop-mediated isothermal amplification.

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