Development of a real-time loop mediated isothermal amplification assay for Stromatinia cepivora in response to an outbreak in Northern Idaho

2021 ◽  
Author(s):  
James Woodhall ◽  
Miranda Harrington ◽  
Lara Brown ◽  
Jennifer Jensen ◽  
Kate Painter
Keyword(s):  
Real Time ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Rapid Development ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
White Rot ◽  
Loop Mediated Isothermal Amplification ◽  
Northern Idaho ◽  
Time Loop

Stromatinia cepivora is the causal agent of white rot disease of Allium species. In 2018, white rot was observed in Boundary county in Northern Idaho in garlic and onion plants in a variety of home and market garden operations. As the university diagnostic lab for Idaho is situated in Parma within a regulated area for Stromatinia cepivora, a point of care (POC) assay using real-time loop mediated isothermal amplification (LAMP) was developed to minimize the amount of material potentially sent to the diagnostic lab. The LAMP assay was used with a BioRanger platform and although the limit of detection was one hundred times less than TaqMan, it was capable of detecting a single sclerotia. This study demonstrates the rapid development and deployment of a POC suitable LAMP assay. Despite limitations in sensitivity and dynamic range compared to real-time PCR, POC LAMP assays are advantageous where biosecurity concerns prohibit the movement of material suitable for diagnosis as well as facilitating engagement with growers.

Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985

2015 ◽  
Vol 98 (5) ◽  
pp. 1207-1214 ◽  
Author(s):  
Gurinder Jit Randhawa ◽  
Rashmi Chhabra ◽  
Rajesh K Bhoge ◽  
Monika Singh
Keyword(s):  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Bt Cotton ◽  
Loop Mediated Isothermal Amplification ◽  
Commercial Cultivation ◽  
Detection And Identification ◽  
Cotton Seeds ◽  
Gm Detection ◽  
Time Loop

Abstract Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

scholarly journals Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5993 ◽  
Author(s):  
Shao-Xin Cai ◽  
Fan-De Kong ◽  
Shu-Fei Xu ◽  
Cui-Luan Yao
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
The Real ◽  
Loop Mediated Isothermal Amplification ◽  
Enterocytozoon Hepatopenaei ◽  
Time Loop

Background Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

scholarly journals Real-time loop-mediated isothermal amplification (LAMP) of mgc2 gene of Mycoplasma gallisepticum

2017 ◽  
Vol 61 (4) ◽  
pp. 439-444 ◽  
Author(s):  
Syed Ehtisham-ul-Haque ◽  
Madiha Kiran ◽  
Usman Waheed ◽  
Muhammad Younus
Keyword(s):  
Real Time ◽  
Gene Sequence ◽  
Molecular Detection ◽  
Lamp Assay ◽  
Mycoplasma Gallisepticum ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Poultry Industry ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

AbstractIntroduction:Mycoplasma gallisepticum is considered the most pathogenic and economically significant avian Mycoplasma spp. for the worldwide poultry industry. The aim of this study was to develop a novel and sensitive real-time loop-mediated isothermal amplification (LAMP) assay based on the amplification of its mgc2 gene sequence for its rapid molecular detection in poultry.Material and Methods: Blood samples from 300 broiler and layer chickens were screened using a rapid serum agglutination (RSA) test. A real-time LAMP reaction was conducted with seropositive swab samples at 60°C for 90 min in an ESEQuant tube scanner using 6-carboxyfluorescein as the reporting dye.Results: The sensitivity of the developed assay was 10 fg/μL of DNA. The assay was found 100% specific, showing no cross-reactivity with other avian Mycoplasma species. The proportion found of the positive samples by the real-time LAMP was 58%. In comparison, the RSA was found to detect 52% of positive cases.Conclusion: The mgc2 real-time LAMP emerged as a more sensitive and accurate method for molecular detection of M. gallisepticum than RSA. Robustness and precision give it applicability as a potential field diagnostic tool for M. gallisepticum control. The study will be beneficial in reducing economic losses that M. gallisepticum inflicts on the poultry industry. This is the first reported development of a real-time LAMP assay based on the amplification of the mgc2 gene sequence using an ESEQuant tube scanner for galline M. gallisepticum detection.

A real-time loop-mediated isothermal amplification for detection of the wheat dwarf virus in wheat and the insect vector Psammotettix alienus

Plant Disease ◽  
2021 ◽  
Author(s):  
Xingan Hao ◽  
Licheng Wang ◽  
Xudong Zhang ◽  
Qinrong Zhong ◽  
Jamal U Ddin Hajano ◽  
...  
Keyword(s):  
Real Time ◽  
Lamp Assay ◽  
High Specificity ◽  
Positive Sample ◽  
Isothermal Amplification ◽  
Dwarf Virus ◽  
Insect Vector ◽  
Wheat Dwarf Virus ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is an economically important and widespread pathogen of cereal crops. It causes huge yield loss in wheat due to the unavailability of resistant varieties and rapid transmission by the vector leafhopper, Psammotettix alienus (Dahlb). To monitor and forecast this viral disease, an early diagnosis method is required for WDV detection in both infected plants and the virus vectors. In this study, we developed a real-time loop-mediated isothermal amplification (LAMP) assay for WDV detection. The positive sample could be detected within 28-32 min by following a simple and cost-effective procedure. The real-time LAMP assay showed a sensitivity of 2.7×105-6 copies/µL for detection and a high specificity for WDV amplification, with a similar accuracy to qPCR. Furthermore, a tube-closed dye method facilitates the inspection of the LAMP reaction and avoids cross contamination in the detection of the virus. This valuable detection assay could serve as an important tool for diagnosis and forecasting wheat dwarf disease intensity in the field.

scholarly journals Rapid And Sensitive Detection Of Lawsonia Intracellularis In Pigs By Real-Time Loop-Mediated Isothermal Amplification

2015 ◽  
Vol 65 (1) ◽  
pp. 20-29 ◽  
Author(s):  
PARK Byung-Yong ◽  
SHIM Kwan-Seob ◽  
KIM Won-Il ◽  
HOSSAIN Md Mukter ◽  
KIM Bumseok ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Detection Methods ◽  
Conventional Pcr ◽  
Fecal Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

Abstract A simple and rapid real-time loop-mediated isothermal amplification (LAMP) assay designed to detect Lawsonia (L.) intracellularis, an important bacteria causing proliferative enteropathy in pigs. A set of four primers targeting the ubiquinone/menaquinone biosynthesis methylase (ubiE) gene was designed for the LAMP reaction. Additionally, serial 10-fold dilutions of cultured L. intracellularis and spiked feces were also used for the optimization of real-time LAMP. The lower limit of the linear range of the assay in L. intracellularis was 1.0 × 100 L. intracellularis. Real-time LAMP was 10 and 100 times more sensitive than real-time PCR and conventional PCR detection methods, respectively. Based on testing of 213 porcine fecal samples using real-time LAMP, realtime PCR and PCR, the agreement quotients of real-time LAMP with conventional PCR and with real-time PCR were 0.77 and 0.95, respectively. This study demonstrated that real-time LAMP was a powerful tool for the rapid and sensitive detection of L. intracellularis in porcine fecal samples.

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