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 A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas Schmidt ◽  
Sandro Berghaus ◽  
Frithjof Blessing ◽  
Folker Wenzel ◽  
Holger Herbeck ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mass Screening ◽  
High Throughput ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification ◽  
Clinical Diagnostic

AbstractShortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen’s kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar’s test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

scholarly journals Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) for COVID-19 Diagnosis: A Systematic Review and Meta-Analysis

2021 ◽  
Author(s):  
Anita Dominique Subali ◽  
Lowilius Wiyono
Keyword(s):  
Systematic Review ◽  
Reverse Transcriptase ◽  
Quality Assessment ◽  
Meta Analysis ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Loop Mediated Isothermal Amplification

ABSTRACTBackgroundCoronavirus Disease 2019 (COVID-19) has caused a severe outbreak and become a global public health priority. Rapid increment of infection number along with significant deaths have placed the virus as a serious threat to human health. Rapid, reliable, and simple diagnostic methods are critically essential for disease control. While Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is the current diagnostic gold standard, Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) appears as a compelling alternative diagnostic test due to its more simplicity, shorter time to result, and lower cost. This study examined RT-LAMP application for rapid identification of SARS-CoV-2 infection compared to RT-PCR assay.MethodsA systematic review and meta-analysis (2020) was conducted in 6 scientific databases following the PRISMA Guideline. Original published studies on human clinical samples in English were included. Articles evaluated sensitivity and specificity of RT-LAMP relative to RT-PCR were considered eligible. Quality assessment of bias and applicability was examined based on QUADAS-2.ResultsA total of 351 studies were found based on the keywords and search queries. 14 eligible case control studies fitted the respective criteria. Quality assessment using QUADAS-2 indicated low risk bias in all included studies. All case studies, comprises 2,112 samples, had the cumulative sensitivity of 95.5% (CI 97.5%=90.8-97.9%) and cumulative specificity of 99.5% (CI 97.5%=97.7-99.9%).ConclusionRT-LAMP assay could be suggested as a reliable alternative COVID-19 diagnostic method with reduced cost and time compared to RT-PCR. RT-LAMP could potentially be utilized during the high-throughput and high-demand critical situations.

Real-Time Reverse Transcriptase PCR for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

2010 ◽  
Vol 73 (3) ◽  
pp. 507-514 ◽  
Author(s):  
CHAYAPA TECHATHUVANAN ◽  
FRANCES ANN DRAUGHON ◽  
DORIS HELEN D'SOUZA
Keyword(s):  
Reverse Transcriptase ◽  
Salmonella Typhimurium ◽  
Real Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Traditional Methods ◽  
Selective Enrichment ◽  
Reverse Transcriptase Pcr

Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 108 to 100 CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37°C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (Tm) analysis to determine specific Salmonella invA (Tm = 87.5°C) and IAC (Tm = 82°C) products. Improved Salmonella detection up to 101 CFU/25 g of pork and 100 CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 106 CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take ≥1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.

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 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.

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.

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.

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