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.

scholarly journals Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (8) ◽  
pp. 2589-2595 ◽  
Author(s):  
Feifei Han ◽  
Fei Wang ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Vibrio Vulnificus ◽  
Lamp Assay ◽  
The United States ◽  
Isothermal Amplification ◽  
Quantitative Detection ◽  
Strain Atcc ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results

ABSTRACTVibrio vulnificusis a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmentalV. vulnificusstrains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study,vcgCwas selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulentV. vulnificusstrains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulentV. vulnificusstrain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 103CFU/g ofV. vulnificusATCC 33815, while showing negative results for a nonvirulentV. vulnificusstrain (515-4c2) spiked at 107CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulentV. vulnificusstrain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulentV. vulnificusstrain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulentV. vulnificusin raw oysters with high speed, specificity, and sensitivity, which may facilitate better control ofV. vulnificusrisks associated with raw oyster consumption.

scholarly journals Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (12) ◽  
pp. 4008-4016 ◽  
Author(s):  
Siyi Chen ◽  
Fei Wang ◽  
John C. Beaulieu ◽  
Rebecca E. Stein ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Sample Treatment ◽  
Propidium Monoazide ◽  
Simple Method ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTRecent outbreaks linked toSalmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targetingSalmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killedSalmonellacells with concentrations up to 108CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viableSalmonellacells in pure culture and 6.1 × 103to 6.1 × 104CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viableSalmonellacell numbers was high (R2= 0.949 to 0.993), with a quantification range (102to 105CFU/reaction in pure culture and 104to 107CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viableSalmonellacells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.

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 The Evolutionary Divergence of Shiga Toxin-Producing Escherichia coli Is Reflected in Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Spacer Composition

2013 ◽  
Vol 79 (18) ◽  
pp. 5710-5720 ◽  
Author(s):  
Shuang Yin ◽  
Mark A. Jensen ◽  
Jiawei Bai ◽  
Chitrita DebRoy ◽  
Rodolphe Barrangou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
False Negative ◽  
The United States ◽  
Evolutionary Divergence ◽  
Content Type ◽  
E Coli ◽  
Qpcr Method ◽  
Flagellar Antigen ◽  
Big Six

ABSTRACTThe Shiga toxin-producingEscherichia coli(STEC) strains, including those of O157:H7 and the “big six” serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature ofclusteredregularlyinterspacedshortpalindromicrepeats (CRISPRs) in phylogenetically relatedE. colistrains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution acrossE. colistrains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.

scholarly journals Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Lu Han ◽  
Kaidi Wang ◽  
Lina Ma ◽  
Pascal Delaquis ◽  
Susan Bach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pure Culture ◽  
Fresh Produce ◽  
Isothermal Amplification ◽  
Propidium Monoazide ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli ◽  
Detection And Quantification

ABSTRACT Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the “viable but nonculturable” (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Detection of Enteroaggregative Escherichia coli by Loop-Mediated Isothermal Amplification

2010 ◽  
Vol 73 (6) ◽  
pp. 1064-1072 ◽  
Author(s):  
EIJI YOKOYAMA ◽  
MASAKO UCHIMURA ◽  
KENITIRO ITO
Keyword(s):  
Escherichia Coli ◽  
Food Samples ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Bacterial Strains ◽  
Exact Test ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Contaminated Food ◽  
Better Than

A novel gene amplification method, loop-mediated isothermal amplification (LAMP), has been recently developed as a rapid, specific diagnostic method for various infectious diseases. We have investigated whether LAMP can be used to detect small numbers of enteroaggregative Escherichia coli (EAEC) cells contaminated in food samples. Primers for LAMP reaction were designed with EAEC aggR gene sequences (available in GenBank). LAMP specificity with these primers was the same as that of PCR in a study of 37 EAEC and 42 non-EAEC bacterial strains. The sensitivity of the LAMP method was better than that of PCR in a study of serially diluted EAEC cells. The LAMP method was significantly more effective than was PCR in detecting EAEC-contaminated food samples (Fisher's exact test, P < 0.05). Therefore, the LAMP method described here should be useful for detecting small numbers of EAEC cells in food samples.

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