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.

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.

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 Rapid detection of Pecan Root-Knot Nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

2020 ◽  
Author(s):  
Sumyya Waliullah ◽  
Jessica Bell ◽  
Tammy Stackhouse ◽  
Ganpati Jagdale ◽  
Abolfazl Hajihassani ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Nematode Species ◽  
Conventional Polymerase Chain Reaction ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Field Conditions ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Root Knot Nematode ◽  
Loop Mediated Isothermal Amplification

AbstractMeloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in yields from mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 65°C, with results 100 times more sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

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 detection of cytochromecd1-containing nitrite reductase encoding genenirSwith loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
Qianqian Yang ◽  
Xuzhi Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification

AbstractThe cytochromecd1-containing nitrite reductase,nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance ofnirSis a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detectnirSgene using those ofPseudomonas aeruginosato optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lackingnirSgene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detectingnirSgene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, thenirSgene ofP. aeruginosain spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

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.

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