scholarly journals Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

2014 ◽  
Vol 80 (8) ◽  
pp. 2516-2525 ◽  
Author(s):  
Fei Wang ◽  
Qianru Yang ◽  
Yinzhi Qu ◽  
Jianghong Meng ◽  
Beilei Ge
Keyword(s):  
Escherichia Coli ◽  
Dna Extraction ◽  
Shiga Toxin ◽  
The United States ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Robust Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Methods

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n= 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 105to 106CFU per 25 g (i.e., 103to 104CFU per g) in produce, except for strains harboring thestx2c,eae-β, andeae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of variousstx2andeaesubtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.

scholarly journals Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anthony Ablordey ◽  
Evans Ahotor ◽  
Charles A. Narh ◽  
Sandra A. King ◽  
Isra Cruz ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Point Of Care ◽  
Buruli Ulcer ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

scholarly journals Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment

2020 ◽  
Vol 18 (3) ◽  
pp. 345-357
Author(s):  
Katherine E. Fisher ◽  
Leah P. Wickenberg ◽  
Lesley F. Leonidas ◽  
Anna A. Ranz ◽  
Michelle A. Habib ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Water Samples ◽  
Traditional Culture ◽  
Culture Method ◽  
The United States ◽  
Extraction Methods ◽  
Waterborne Pathogen ◽  
Legionnaires Disease ◽  
Dna Extraction Methods ◽  
Validation Testing

Abstract The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10–14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

scholarly journals Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification

2012 ◽  
Vol 78 (8) ◽  
pp. 2727-2736 ◽  
Author(s):  
Fei Wang ◽  
Lin Jiang ◽  
Qianru Yang ◽  
Witoon Prinyawiwatkul ◽  
Beilei Ge
Keyword(s):  
Escherichia Coli ◽  
Pure Culture ◽  
False Negative ◽  
Ground Beef ◽  
The United States ◽  
Food Samples ◽  
Isothermal Amplification ◽  
Specific Detection ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTEscherichia coliO157 and six additional serogroups of Shiga toxin-producingE. coli(STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (wzxorwzy) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 103to 104CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing.

Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in 375 Grams of Beef Trim Enrichments across Multiple Commercial PCR Detection Platforms

2015 ◽  
Vol 78 (1) ◽  
pp. 196-202 ◽  
Author(s):  
SARITA RAENGPRADUB WHEELER ◽  
PRECIAUS HEARD ◽  
CHRISTOPHE DUFOUR ◽  
DELPHINE THEVENOT-SERGENTET ◽  
ESTELLE LOUKIADIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Limit Of Detection ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Detection Methods ◽  
Primary Concern ◽  
The European Union ◽  
Commercial Method

Although serotype O157:H7 remains the pathogenic Shiga toxin–producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

scholarly journals Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing

2020 ◽  
Vol 6 (2) ◽  
pp. 36 ◽  
Author(s):  
Miyono M. Hendrix ◽  
Carla D. Cuthbert ◽  
Suzanne K. Cordovado
Keyword(s):  
Cystic Fibrosis ◽  
Next Generation Sequencing ◽  
Newborn Screening ◽  
Dna Extraction ◽  
Technical Assistance ◽  
The United States ◽  
Extraction Methods ◽  
Next Generation ◽  
Dna Extraction Methods ◽  
Generation Sequencing

An increasing number of newborn screening laboratories in the United States and abroad are moving towards incorporating next-generation sequencing technology, or NGS, into routine screening, particularly for cystic fibrosis. As more programs utilize this technology for both cystic fibrosis and beyond, it is critical to identify appropriate DNA extraction methods that can be used with dried blood spots that will result in consistent, high-quality sequencing results. To provide comprehensive quality assurance and technical assistance to newborn screening laboratories wishing to incorporate NGS assays, CDC’s Newborn Screening and Molecular Biology Branch designed a study to evaluate the performance of nine commercial or laboratory-developed DNA extraction methods that range from a highly purified column extraction to a crude detergent-based no-wash boil prep. The DNA from these nine methods was used in two NGS library preparations that interrogate the CFTR gene. All DNA extraction methods including the cruder preps performed reasonably well with both library preps. One lower-concentration, older sample was excluded from one of the assay evaluations due to poor performance across all DNA extraction methods. When 84 samples, versus eight, were run on a flow cell, the DNA quality and quantity were more significant variables.

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