scholarly journals Development of a Real-Time Loop-Mediated Isothermal Amplification Method for the Detection of West Nile Virus

2020 ◽  
Vol 13 (10) ◽  
Author(s):  
Jae Woong Lee ◽  
Yu-Jung Won ◽  
Sung-Geun Lee ◽  
Soon-Young Paik
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Genomic Sequences ◽  
The Novel ◽  
Rt Pcr ◽  
West Nile ◽  
The Real ◽  
E Gene

Background: The West Nile Virus (WNV), discovered in New York, USA in 1999 after it was first isolated in Uganda in 1937, has since spread not only in the United States but also around the world. Africa, Eurasia, Australia, and the Middle East have sporadic cases of the disease. Objectives: We aimed to find real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to be more sensitive than conventional RT-PCR, and more rapid and efficient than conventional RT-PCR and real-time RT-PCR for WNV detection. Methods: A total of 32 genomic sequences from different strains of WNV were analyzed to identify conserved nucleotide sequence regions. Six WNV specific RT-LAMP primers targeting the E gene were designed. Results: The novel primer for the real-time RT-LAMP assay can detect WNV with high specificity. The efficiency of the real-time RT-LAMP assay is higher than the conventional RT-PCR and real-time RT-PCR. Real-time RT-PCR and conventional PCR require at least 30 – 40 min and 2 h, respectively, to yield results, whereas real-time RT-LAMP provides positive results in only 10 – 20 min. Conclusions: The novel primers were developed by analyzing of 32 genomic sequences of WNV strains. The primers were designed from the most conserved region of the E gene for real-time RT-LAMP. The LAMP assay is a rapid, efficient, highly sensitive, and specific tool for the identification of WNV.

scholarly journals Laboratory and Field Evaluations of a Commercially Available Real-Time Loop-Mediated Isothermal Amplification Assay for the Detection of West Nile Virus in Mosquito Pools

2021 ◽  
Vol 37 (4) ◽  
pp. 256-262
Author(s):  
Kristen L. Burkhalter ◽  
Michael O'Keefe ◽  
Zachary Holbert-Watson ◽  
Theodore Green ◽  
Harry M. Savage ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Vector Surveillance ◽  
West Nile ◽  
Loop Mediated Isothermal Amplification ◽  
Surveillance Programs ◽  
Kappa Agreement

ABSTRACT Although the specific cDNA amplification mechanisms of reverse-transcriptase polymerase chain reaction (RT-PCR) and RT loop-mediated isothermal amplification (RT-LAMP) are very different, both molecular assays serve as options to detect arboviral RNA in mosquito pools. Like RT-PCR, RT-LAMP uses a reverse transcription step to synthesize complementary DNA (cDNA) from an RNA template and then uses target-specific primers to amplify cDNA to detectable levels in a single-tube reaction. Using laboratory-generated West Nile virus (WNV) samples and field-collected mosquito pools, we evaluated the sensitivity and specificity of a commercially available WNV real-time RT-LAMP assay (Pro-AmpRT™ WNV; Pro-Lab Diagnostics, Inc., Round Rock, Texas) and compared the results to a validated real-time RT-PCR assay. Laboratory generated virus stock samples containing ≥ 2.3 log10 plaque-forming units (PFU)/ml and intrathoracically inoculated mosquitoes containing ≥ 2.4 log10 PFU/ml produced positive results in the Pro-AmpRT WNV assay. Of field-collected pools that were WNV positive by real-time RT-PCR, 74.5% (70 of 94) were also positive by the Pro-AmpRT WNV assay, resulting in an overall Cohen's kappa agreement of 79.4% between the 2 tests. The Pro-AmpRT WNV assay shows promise as a suitable virus screening tool for vector surveillance programs provided agencies are aware of its characteristics and limitations.

Establishment and evaluation of real-time PCR for West Nile virus detection

2009 ◽  
Vol 6 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Shi Li-Jun ◽  
Lu Mao-Min ◽  
Li Gang ◽  
Li Cheng-Yao ◽  
Zhang Jin-Gang
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Virus Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
West Nile ◽  
Capsid Protein Gene ◽  
Specific Test ◽  
Intercalating Dye ◽  
Polymerase Chain

AbstractA rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Alternaria alternata

2017 ◽  
Vol 100 (1) ◽  
pp. 99-103 ◽  
Author(s):  
Xinyue Zhang ◽  
Guojie Xu ◽  
Huaqi Tang ◽  
Yanpeng Li ◽  
Chunsheng Liu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Alternaria Alternata ◽  
Lamp Assay ◽  
High Specificity ◽  
Etiologic Agent ◽  
Isothermal Amplification ◽  
Pcr Assay ◽  
The Real ◽  
Loop Mediated Isothermal Amplification

Abstract Fungi of the Alternaria genus are associated with allergic diseases, with Alternaria alternata being one of the most prevalent species. A. alternata has been frequently reported as the etiologic agent of hypersensitivity pneumonitis, allergic rhinosinusitis, bronchial asthma,and other diseases. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay and a real-time PCR assay to detect low levels of A. alternata in herbal tea samples. The LAMP assay can detect as little as 3 pg/μL of A. alternata genomic DNA with high specificity. In addition, both the LAMP assay and the real-time PCR assay can be used for quantification of A. alternata. Although the newly developed LAMP assay is more rapid and specific in A. alternata identification, the real-time PCR assay is more precise in quantitation analysis.

A novel quantitative multiplex real-time RT-PCR for the simultaneous detection and differentiation of West Nile virus lineages 1 and 2, and of Usutu virus

2013 ◽  
Vol 189 (2) ◽  
pp. 321-327 ◽  
Author(s):  
Javier Del Amo ◽  
Elena Sotelo ◽  
Jovita Fernández-Pinero ◽  
Carmina Gallardo ◽  
Francisco Llorente ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
West Nile ◽  
Usutu Virus

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 RT-PCR Assays for Detection and Genotyping of West Nile Virus Lineages Circulating in Africa

2016 ◽  
Vol 16 (12) ◽  
pp. 781-789 ◽  
Author(s):  
Gamou Fall ◽  
Martin Faye ◽  
Manfred Weidmann ◽  
Marco Kaiser ◽  
Anne Dupressoir ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Real Time ◽  
Rt Pcr ◽  
West Nile ◽  
Pcr Assays

Comparison of Reverse Transcriptase PCR, Reverse Transcriptase Loop-Mediated Isothermal Amplification, and Culture-Based Assays for Salmonella Detection from Pork Processing Environments

2011 ◽  
Vol 74 (2) ◽  
pp. 294-301 ◽  
Author(s):  
CHAYAPA TECHATHUVANAN ◽  
FRANCES ANN DRAUGHON ◽  
DORIS HELEN D'SOUZA
Keyword(s):  
Reverse Transcriptase ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Water Bath ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Loop Mediated Isothermal Amplification

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37°C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62°C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I–based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry.

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