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.

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.

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.

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 Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

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 and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

2005 ◽  
Vol 54 (11) ◽  
pp. 1037-1041 ◽  
Author(s):  
Ryoichi Saito ◽  
Yoshiki Misawa ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
Kimiko Ubukata ◽  
...  
Keyword(s):  
Real Time ◽  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Clinical Laboratory ◽  
Direct Sequencing ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

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