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

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Fei Wang, Qianru Yang, Yinzhi Qu, Jianghong Meng and Beilei Ge Appl. Environ. Microbiol. 2014, 80(8):2516. DOI: 10.1128/AEM.04203-13. Published Ahead of Print 7 February 2014.

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid, Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce Fei Wang,a,b Qianru Yang,a,c Yinzhi Qu,b Jianghong Meng,b Beilei Gea

Shiga toxin-producing Escherichia 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 ⴝ 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 105 to 106 CFU per 25 g (i.e., 103 to 104 CFU per g) in produce, except for strains harboring the stx2c, eae-␤, and eae-␪ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes 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.

S

higa toxin-producing Escherichia coli (STEC) strains are zoonotic agents of significant public health concern (1). In the United States, STEC ranked among the top three causes of foodborne disease outbreaks as well as outbreak-associated illnesses, hospitalizations, and deaths from 1998 to 2010 (2, 3). Although E. coli O157:H7 remains the single most common STEC strain (4), the clinical importance of certain non-O157 STEC strains is growing worldwide (5). Recent years of FoodNet data in the United States have consistently shown more laboratory-confirmed infections caused by non-O157 STEC strains than O157 STEC strains (6–8). Currently, the U.S. regulation designates seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, nonintact beef products (9). Produce ranked second only to beef in causing the largest percentage of STEC outbreaks, many of which are large-scale, multistate outbreaks (3). In September 2006, tainted prepackaged spinach triggered an E. coli O157:H7 outbreak, resulting in 205 confirmed illnesses and 3 deaths in 26 states (10). In 2010, a multistate outbreak of STEC O145 infections linked to shredded romaine lettuce from a single processing facility led to 26 confirmed cases in five states (11). The May 2011 massive outbreak of hemolytic-uremic syndrome (HUS; 852 cases) in Germany and several other countries was attributed to a rare STEC serotype, serotype O104:H4, in sprouts (12). Additionally, several recent multistate outbreaks have been caused by E. coli O157:H7 in romaine lettuce, organic spinach/spring mix blend, and ready-to-eat salads and by STEC O26 in clover sprouts (13). To reduce the incidence of produce-associated outbreaks, a multifaceted approach from farm to table is required. In particu-

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lar, the industry has drastically increased raw and finished product testing as a tool to better identify contamination risks (14). Nonetheless, STEC detection in produce remains a challenging task (15, 16). Due to the highly perishable nature of produce, a rapid test is critical. Produce items are also diverse and complex, with many harboring assay inhibitors and therefore requiring effective sample preparation and commodity-specific method validation (16). Additionally, pathogens in produce are usually injured cells present at low levels, whereas the normal flora is present at high levels, resulting in the requirement for a highly sensitive and specific assay (15). The need to identify STEC as a group and certain STEC serogroups specifically adds yet another layer of complexity (17). Owing to their rapidity, specificity, and sensitivity, molecularbased methods, such as PCR and real-time quantitative PCR (qPCR), have gained widespread applications in produce testing (14, 16). Enrichment is commonly used to increase target cell numbers and simultaneously dilute assay inhibitors and the normal flora in produce (15). However, false-positive and false-neg-

Received 18 December 2013 Accepted 5 February 2014 Published ahead of print 7 February 2014 Editor: D. W. Schaffner Address correspondence to Beilei Ge, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.04203-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.04203-13

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Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, Maryland, USAa; Department of Nutrition and Food Science, University of Maryland, College Park, Maryland, USAb; Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, USAc

LAMP Assays for STEC in Produce

MATERIALS AND METHODS Bacterial strains and target gene characterization. A total of 123 E. coli strains representing 41 serogroups and 33 non-E. coli strains (Table 1) were used for specificity testing. Among them, seven STEC strains (underlined in Table 1), one from each of the seven adulterant serogroups, were used for sensitivity testing and spiked-produce experiments. All strains were cultured as described previously (33). E. coli strains were examined for the presence of STEC virulence genes (stx1, stx2, and eae) by PCR, followed by restriction fragment length polymorphism (RFLP) analysis to determine their respective gene subtypes (36, 37). LAMP. A LAMP suite of 10 assays recently developed by our research group (32, 33) was evaluated. The first three assays targeted common STEC virulence genes (stx1, stx2, and eae), while the remaining seven each targeted a gene (wzx or wzy) on the O-antigen gene clusters of the seven adulterant STEC serogroups. Each LAMP assay employed five to six specially designed LAMP primers (see Table S1 in the supplemental material), two outer primers, two inner primers, and one or two loop primers that recognized specific regions of the target DNA sequences. The assays were performed as described previously (32, 33). Briefly, the LAMP reagent mix (25 ␮l) contained 1⫻ ThermoPol reaction buffer (New England BioLabs, Ipswich, MA), 6 mM MgSO4, 1.2 mM each deoxynucleoside triphosphate (dNTP), 0.1 ␮M each outer primer (Integrated DNA Technologies, Coralville, IA), 1.8 ␮M each inner primer, 1

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␮M each loop primer, 10 U of Bst DNA polymerase (New England BioLabs), and 2 ␮l of template DNA. All LAMP reactions were carried out at 65°C (63°C for the O157 LAMP) for 1 h and terminated at 80°C for 5 min in an LA-320C real-time turbidimeter (Eiken Chemical Co., Ltd., Tokyo, Japan). Turbidity readings at 650 nm were obtained every 6 s, and time threshold (Tt) values (in minutes) were determined when the turbidity increase measurements (differential values of the moving average of the turbidity) exceeded 0.1. qPCR. As a comparison, qPCR assays developed by USDA scientists (34, 35) were performed. Similarly, 10 sets of primers/probes were used; 3 targeted common STEC virulence genes (stx1, stx2, and eae) and 7 targeted the wzx or wzy gene on the O-antigen gene clusters of the seven adulterant STEC serogroups. The qPCR assays were carried out as described previously (32, 33). The reagent mix (25 ␮l) consisted of 1⫻ PCR buffer, 4 mM MgCl2, 0.2 mM each dNTP, 0.25 ␮M each primer (see Table S1 in the supplemental material), 0.1875 ␮M probe, 1.5 U of GoTaq Hot Start polymerase (Promega, Madison, WI), and 2 ␮l of template DNA. The qPCRs were conducted using 40 cycles of denaturation at 94°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 50 s in a SmartCycler II system (Cepheid, Sunnyvale, CA). Fluorescence readings were acquired using the 6-carboxyfluorescein (FAM) channel, and cycle threshold (CT) values (in number of cycles, with approximately 2 min per cycle) were recorded when the fluorescence readings reached 30 units. Specificity and sensitivity tests. For assay specificity, DNA templates of the 156 bacterial strains (Table 1) were prepared by heating at 95°C for 10 min. Aliquots (2 ␮l) were subjected to LAMP and qPCR, and assays were repeated twice. Assay sensitivity (limit of detection) was determined by using 10-fold serial dilutions of the seven STEC strains of the adulterant serogroups. DNA templates were prepared from stationary-phase cultures as described previously (32). Aliquots (2 ␮l) were tested by LAMP and qPCR, and assays were repeated three times. Assay evaluation with produce with high-level inoculation (assay sensitivity for detection of STEC in produce). Eight produce items, including varieties of lettuce (iceberg, romaine), spinach (baby, savoy, semisavoy), and sprouts (alfalfa, clover, mung bean), were obtained from a local grocery store and analyzed within 2 h of collection. Briefly, lettuce and spinach leaves were cut into 4-cm2 pieces using sterile scissors, and 25-g samples were weighed out. Sprouts were also divided into 25-g analysis portions. To determine assay sensitivity for detection of STEC in produce, for each produce item, 35 samples (one sample per strain [n ⫽ 7] per inoculation level [n ⫽ 5]) were inoculated and 3 samples were included as uninoculated controls. Spot inoculation on the produce surface was performed as described previously (38). Briefly, 1.5 ml of overnight STEC cultures 10-fold serially diluted from 105 to 109 CFU/ml was added to each 25-g test sample. For lettuce and spinach, the inoculum was equally divided among the number of leaf pieces. Sprout samples were grouped into three equal portions, and 500 ␮l of the inoculum was added onto the surface of each portion. Aerobic plate counts were determined for the uninoculated controls (n ⫽ 3) by the standard pour plate method. All samples were air dried in a laminar flow biosafety cabinet for 2 h, followed by storage at 4°C for 48 h. After cold storage, each sample was homogenized with 225 ml of buffered peptone water (BD Diagnostic Systems, Sparks, MD) for 1 min in a food stomacher (model 400; Seward, Cincinnati, OH). Aliquots (1 ml) of each homogenate were centrifuged at 16,000 ⫻ g for 3 min, and pellets were suspended in 100 ␮l of PrepMan Ultra sample preparation reagent (Applied Biosystems, Foster City, CA). The mixtures were heated at 95°C for 10 min and centrifuged again at 12,000 ⫻ g for 2 min. The supernatants (2 ␮l) were tested by both LAMP and qPCR, and assays were repeated three times. Assay evaluation with produce with low-level inoculation. For assay evaluation with produce with low-level inoculation, the same eight pro-

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ative results are observed, and few PCR assays for STEC have been validated on a commodity-specific basis (16). Besides, a sophisticated thermal cycling instrument is indispensable to carry out these nucleic acid amplification tests (NAATs), limiting their wider applications. Recently, a novel NAAT termed loop-mediated isothermal amplification (LAMP) has emerged as a promising alternative to PCR for pathogen detection (18, 19). LAMP uses four to six specially designed primers and a strand-displacing Bst DNA polymerase to amplify up to 109 copies of target DNA under isothermal conditions (⬃65°C) within an hour (19). Since it is isothermal, LAMP can be performed in much simpler instruments, such as a heater or water bath. To date, multiple LAMP assays targeting STEC Shiga toxin genes (stx1 and stx2) have been developed (20– 24) and evaluated in food, primarily beef (25–28), as have several others targeting the E. coli O157 rfbE gene (encoding perosamine synthetase) (23, 24, 29–31). Very recently, we developed a suite of LAMP assays for STEC (targeting common virulence genes stx1, stx2, and eae) and the seven adulterant STEC serogroups (targeting the wzx or wzy gene on the respective O-antigen gene clusters) (32, 33). Although it has been reported to be rapid, specific, and sensitive, the LAMP suite has not been evaluated using a large number of STEC strains harboring various stx and eae subtypes or tested with a variety of produce items using conditions mimicking real-world contamination events (e.g., low-level surface inoculation, cold storage). In addition, despite the critical role that sample preparation plays, there is a scarcity of data regarding the effectiveness of DNA extraction methods for STEC detection in produce. The aims of this study were to comprehensively evaluate the STEC LAMP suite against qPCR using a large panel of bacterial strains and various produce items (varieties of lettuce, spinach, and sprouts) under conditions mimicking real-world contamination events and to examine the effect of DNA extraction methods on assay performance. The qPCR assays tested were recently developed by scientists from the U.S. Department of Agriculture (USDA) (34, 35).

Wang et al.

TABLE 1 Bacterial strainsc used in this study to evaluate the specificity and sensitivity of the LAMP suite and qPCR assays Strain group STEC strains of target serogroups (n ⫽ 83) O26 (n ⫽ 20)

O103 (n ⫽ 20)

O111 (n ⫽ 19)

Straina

O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O26:H11 O45:H2 O45:H2 O45:H2 O45:H2 O45:H2 O45:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H2 O103:H11 O103:H11 O103:H25 O103:H25 O103:H25 O111:H8 O111:H8 O111:H8 O111:H8 O111:H8 O111:H8 O111:H8 O111:H8 O111:H11 O111:H11 O111:H11 O111:H11

CVM 9935 CVM 9942 CVM 9952 CVM 9953 CVM 9965 CVM 9966 CVM 9967 CVM 9988 CVM 9995 CVM 9997 CVM 9998 CVM 9999 CVM 10000 CVM 10001 CVM 10007 CVM 10008 CVM 10112 CVM 10128 CVM 10224 SJ3 05-6545 A9619-C2 EC1467 EC1674 MI4 SJ9 7828/95 CVM 9260 CVM 9301 CVM 9305 CVM 9318 CVM 9322 CVM 9328 CVM 9380 CVM 9385 CVM 9439 CVM 9440 CVM 9446 CVM 9451 CVM 9453 PMK-5 CVM 9320 SJ12 CVM 9340 CVM 9353 CVM 9354 CVM 9467 CVM 9557 CVM 9574 CVM 9603 CVM 9610 CVM 9614 CVM 9617 CVM 9619 CVM 9505 CVM 9529 CVM 9530 CVM 9534

stx1 subtype

stx2 subtype

stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx2a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a

stx2a stx2a

stx2d

stx2d stx2a

Intimin subtype

Origin

Sourceb

␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ␤ ε ε ε ε ε ␤ ε ε ε ε ε ε ε ε ε ε ε ε ε ε ε ␤ ␤ ␪ ␪ ␪ ␪ ␪ ␪ ␪ ␪ ␪ ␪ ␪ ␤ ␤ ␤ ␤

Animal (antelope) Animal (cow) Animal (pig) Animal (pig) Animal (cow) Animal (cow) Animal (cow) Animal (cow) Human Human Human Human Human Human Human Human Animal (cow) Human Human Unknown Unknown Unknown Human Human Unknown Unknown Human Animal (deer) Animal (goat) Animal (sheep) Animal (cow) Animal (cow) Human Human Human Human Human Human Human Human Human Animal (cow) Unknown Human Animal (cow) Animal (cow) Animal (cow) Animal (cow) Human Human Animal (cow) Animal (cow) Animal (cow) Human Animal (cow) Animal (cow) Animal (pig) Animal (cow)

FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM CDC Lab collection Lab collection FDA, CFSAN FDA, CFSAN Lab collection CDC Lab collection FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM Lab collection FDA, CVM CDC FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM FDA, CVM

(Continued on following page)

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O45 (n ⫽ 6)

Serotype or species

LAMP Assays for STEC in Produce

TABLE 1 (Continued) Strain group

O121 (n ⫽ 3)

O157 (n ⫽ 10)

Other STEC strains (n ⫽ 25)

Other E. coli strains (n ⫽ 15)

Straina

O111:H11 O111:H11 O111:H11 O111:H11 O111:NM O111:NM O111:NM O121:H19 O121:NM O121:NM O145:H28 O145:H28 O145:H28 O145:NM O145:NM O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7 O157:H7

CVM 9535 CVM 9548 CVM 9553 CVM 9591 78/92 P1340 SJ13 ESC0601 EC1406 EC1670 CVM 9785 CVM 9790 EC1792 EC1789 SJ23 85-1 B6903 C8 E-0019 E-0122 E-0342 EC600V MDL 3562 OH-495-189 W2-2

O2:H27 O5:NM O5:NM O8:H28 O15:H16 O22:H8 O36:H14 O46:H38 O50:H7 O55:H7 O73:H18 O83:H8 O88:H49 O91:H21 O104:H21 O113:H21 O116:H21 O125:NM O126:H8 O128:H16 O146:H21 O168:H8 O174:H36 ONT:H7 OX25

SJ5 3143-85 3812-3 ESC0604 N5789 P1330 ESC0603 P1332 3056-85 5906 ESC0608 N11682 P1333 P1334 94-3024 ESC0615 ESC0609 3153-86 78-4084 CVM 9652 90–3158 ESC0613 ESC0602 N5545 ESC0606

O1:K1:H7 O3:K2ab:H2 O9:K103 O18:K1:H7 O44:K74:H18 O77:K96:NM O78:H11 O86:H25 O111:H11

U5-41 U414-41 987 88-766 H702c E10 EC463 H35A CVM 9515

stx1 subtype stx1a stx1a stx1a stx1a stx1a stx1a stx1a

stx2 subtype

stx2a stx2a stx2a stx2a stx2a

stx1a stx1a stx1a stx1a stx1a stx1a stx1a stx1a

stx1a

stx2c stx2a stx2a stx2a stx2a stx2a stx2a stx2a stx2c stx2a stx2a stx2a stx2a stx2a

stx1a stx1c

stx1a

stx1a

stx2a stx2g stx2d stx2g stx2d stx2a stx2d stx2a stx2d stx2a stx2d stx2a stx2a stx2d

stx1a stx1a stx1a stx1a

Intimin subtype

Origin

Sourceb

␤ ␤ ␤ ␤ ␪ ␪ ␪ ε ε ε ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1 ␥1

Animal (cow) Animal (cow) Animal (cow) Animal (cow) Human Animal (calf) Unknown Food (lettuce) Human Unknown Animal (Cow) Human Unknown Human Unknown Human Human Animal (sheep) Animal (calf) Animal (calf) Animal (calf) Food (steak) Food (produce) Food (beef) Food (poultry)

FDA, CVM FDA, CVM FDA, CVM FDA, CVM Lab collection Lab collection CDC FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CVM FDA, CVM FDA, CFSAN FDA, CFSAN CDC Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection

Unknown Unknown Animal (sheep) Food (lettuce) Food (beef) Food (beef) Food (sprouts) Food (beef) Unknown Unknown Food (spinach) Food (beef) Food (meat) Animal (cow) Unknown Food (spinach) Food (spinach) Unknown Unknown Animal (okapi) Unknown Food (spinach) Food (lettuce) Food (beef) Food (spinach)

CDC Lab collection Lab collection FDA, CFSAN Lab collection Lab collection FDA, CFSAN Lab collection Lab collection Lab collection FDA, CFSAN Lab collection Lab collection Lab collection Lab collection FDA, CFSAN FDA, CFSAN Lab collection Lab collection FDA, CVM Lab collection FDA, CFSAN FDA, CFSAN Lab collection FDA, CFSAN

Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Animal (cow)

Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection FDA, CVM

␤

ε ␥1

␤

stx2a stx2d stx2d stx2d

␤

(Continued on following page)

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O145 (n ⫽ 5)

Serotype or species

Wang et al.

TABLE 1 (Continued) Strain group

Straina

O118:H16 O124:NM O145 O145:NM O145:NM O157:H7

P1341 EC230 CVM 9818 EC1790 EC1793 G-13

ATCC 33559 ATCC 43430 ATCC 35222 10053 95MV2 ATCC 23280 ATCC 19120 ATCC 33090 ATCC 19119 ATCC 15313 UMD 489 ATCC 35897 FDA71 SE 9 63B2 7N 37 N 1H 1501 H FDA197 1410 H 2861 H 1243 H ATCC 43971 RH12-23-11 RH12-25-9 G45 ATCC 35048 15937-E6 ATCC 35084 ATCC 33655 ATCC 27969 515-4C2

stx1 subtype

stx2 subtype

Intimin subtype

␥1 ␥1

Origin

Sourceb

Animal (calf) Unknown Animal (cow) Unknown Unknown Animal (sheep)

Lab collection Lab collection FDA, CVM FDA, CFSAN FDA, CFSAN Lab collection

Animal (pig) Animal (calf) Animal (dog) Unknown Human Unknown Animal Animal (cow) Animal (sheep) Animal (rabbit) Unknown Plant Unknown Unknown Unknown Unknown Food (candy) Food (whole eggs) Feed (feather meal) Unknown Feed (feather meal) Animal (turtle) Feed (bonemeal) Unknown Unknown Unknown Unknown Animal (oyster) Unknown Animal (shark) Human Animal (crab) Animal (oyster)

Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN FDA, CFSAN Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection

a

The seven underlined strains were used for the evaluation of specificity and sensitivity and spiked-produce experiments, while the others were used only for the specificity testing. CDC, Centers for Disease Control and Prevention; FDA, CFSAN, U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition; FDA, CVM, U.S. Food and Drug Administration, Center for Veterinary Medicine; Lab collection, the strain collection maintained at the University of Maryland, College Park, MD. c A total of 156 strains were tested. b

duce items described above were used and similar inoculation, processing, and testing procedures were followed, with three major exceptions: inoculation level, enrichment, and replication scheme. The inoculation level applied was 100 CFU/25 g, with two additional ones (102 and 103 CFU/25 g) being used for sprouts only. After surface inoculation and cold storage, the samples were first incubated at 42°C for up to 24 h, and aliquots (1 ml) were removed at 6, 8, 10, and 24 h for processing by use of the PrepMan Ultra reagent and testing by LAMP and qPCR as described above. The experiments were independently repeated three times with different produce samples. In total, there were 21 inoculated samples (one sample per strain [n ⫽ 7] in three repeats [n ⫽ 3]) and 3 uninoculated controls tested per produce item. The number of inoculated samples was tripled for the sprouts varieties, i.e., 63 samples were tested, due to the two additional inoculation levels tested. Comparison of DNA extraction methods. Six DNA extraction methods were evaluated using the same eight produce items described above

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spiked with 1.2 to 1.8 CFU/25 g (or 1.2 ⫻ 102 to 1.8 ⫻ 102 CFU/25 g for sprouts varieties) of E. coli O157:H7 strain MDL 3562 and enriched for various periods (6, 8, 10, 24 h). For each produce item, experiments were independently repeated three times with different samples. These methods were (i) testing of raw enrichment broth, i.e., direct testing without any DNA preparation step; (ii) boiling preparation at 95°C for 10 min; (iii) two-step centrifugation by first centrifuging at 500 ⫻ g for 1 min to remove produce tissues and then centrifuging again at 16,000 ⫻ g for 5 min to pellet bacterial cells and resuspending the pellet in 100 ␮l of TE (Tris-EDTA) buffer; (iv) two-step centrifugation followed by boiling, i.e., heating the bacterial suspension in TE buffer prepared by the two-step centrifugation method at 95°C for 10 min; (v) the method with the PrepMan Ultra reagent, as describe above; and (vi) the FTA Elute method, which, briefly, consisted of adding 65 ␮l of enrichment broths onto an FTA card (Whatman Inc., Florham Park, NJ), punching out two 2-mm disks after absorption, allowing the disks to dry, washing the disks with

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Other bacteria (n ⫽ 33) Campylobacter coli Campylobacter jejuni Campylobacter lari Citrobacter freundii Enterobacter cloacae Hafnia alvei Listeria grayi Listeria innocua Listeria ivanovii Listeria monocytogenes Listeria seeligeri Listeria welshimeri Salmonella enterica serovar Braenderup S. enterica serovar Enteritidis S. enterica serovar Heidelberg S. enterica serovar Javiana S. enterica serovar Mbandaka S. enterica serovar Montevideo S. enterica serovar Muenchen S. enterica serovar Newport S. enterica serovar Oranienburg S. enterica serovar Poona S. enterica serovar Stanley S. enterica serovar Typhimurium Shigella boydii Shigella dysenteriae Shigella flexneri Vibrio aestuarianus Vibrio cholerae Vibrio harveyi Vibrio mimicus Vibrio parahaemolyticus Vibrio vulnificus

Serotype or species

LAMP Assays for STEC in Produce

TABLE 2 Sensitivity of the LAMP suite and qPCR assays in pure culture and with spiked producek Detection limit (CFU/reaction in pure culture or CFU/25 g in spiked produce) Target gene and subtype

Stx1 Stx2 Stx2 Stx2 Intimin Intimin Intimin Intimin O26 O45 O103 O111 O121 O145 O157

stx1a stx2a stx2c stx2d eae-␤ eae-␥1 eae-ε eae-␪ wzy (O26) wzy (O45) wzx (O103) wzy (O111) wzy (O121) wzx (O145) wzy (O157)

Pure culture

Lettuce

Spinach

Sprouts

Strain

LAMP

qPCR

LAMP

qPCR

LAMP

qPCR

LAMP

qPCR

SJ9 SJ9 CVM 9790 CVM 9322 SJ9 CVM 9790 CVM 9322 SJ13 SJ3 SJ9 CVM 9322 SJ13 ESC0601 CVM 9790 MDL 3562

10 1–10b 1 ⫻ 103 14 1 ⫻ 102 10 14 2 ⫻ 102 12 1–10a 14 20 20 10 12

10 10 1–10b 14 10 1–10b 14 2–20a 1.2–12a 1–10b 14 2 20 10 1.2–12a

1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 107 1.5 ⫻ 105 1.4 ⫻ 106-107c 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 108 1.5 ⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.4 ⫻ 105

1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.5⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.4 ⫻ 105

1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 107 1.5 ⫻ 105 1.4 ⫻ 107 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 108 1.5 ⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105-106d 1.4 ⫻ 105 1.7 ⫻ 105 1.4 ⫻ 105

1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105-106e 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105-106f 1.5 ⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.4 ⫻ 105

1.4 ⫻ 105-106g 1.4 ⫻ 105 1.7 ⫻ 107 1.5 ⫻ 105-106h 1.4 ⫻ 107 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 108 1.5 ⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105-106g 1.4 ⫻ 105 1.7 ⫻ 105-106g 1.4 ⫻ 105

1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.5 ⫻ 105-106g 1.4 ⫻ 105-106i 1.7 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105-106j 1.5 ⫻ 105 1.4 ⫻ 105 1.5 ⫻ 105 1.4 ⫻ 105 1.4 ⫻ 105 1.7 ⫻ 105 1.4 ⫻ 105

a

One out of three repeats was positive for the lower detection limit. Two out of three repeats were positive for the lower detection limit. c Iceberg lettuce had the lower detection limit. d Baby and savoy spinach had the lower detection limit. e Baby spinach had the lower detection limit. f Savoy and semisavoy spinach had the lower detection limit. g Clover and mung bean sprouts had the lower detection limit. h Clover sprouts had the lower detection limit. i Alfalfa and clover sprouts had the lower detection limit. j Alfalfa and mung bean sprouts had the lower detection limit. k In produce testing, 1.5 ml of 10-fold serially diluted overnight STEC cultures (prepared at different times from those used in the pure culture testing) was added to each 25-g produce sample. b

sterile water, and then heating with water at 95°C for 30 min. Aliquots (2 ␮l) of DNA templates prepared by all six methods were tested by eae LAMP and eae qPCR. Data analysis. Means and standard deviations of Tt values for LAMP and CT values for qPCR were calculated by use of the Microsoft Excel program (Seattle, WA). The Tt and CT values sorted by assay target, gene subtype, produce type, enrichment time, and DNA extraction method were compared by using analysis of variance (SAS for Windows, version 9.2; SAS Institute, Cary, NC). Differences between the mean values were considered significant when P was ⬍0.05.

RESULTS

Assay specificity. Among 156 bacterial strains (Table 1) tested by the STEC LAMP suite, false-positive or false-negative results were not observed; i.e., LAMP results matched 100% with known strain characteristics for all of the target genes. The overall mean Tt values ranged from 16.1 min for eae LAMP to 24.3 min for stx2 LAMP (data not shown). Among the three assays targeting STEC virulence genes, stx1 and eae LAMPs yielded significantly lower Tt values than stx2 LAMP, while O26 and O121 LAMPs had the lowest Tt values among the seven serogroup-specific assays (P ⬍ 0.05) (data not shown). The Tt values also differed among strains carrying various stx2 and eae subtypes. Notably, the stx2 LAMP proceeded the slowest in strains harboring stx2g (mean Tt, 30.6 min), followed by LAMPs with stx2c (24.7 min), stx2d (24.4 min), and stx2a (23.7 min), while by the eae LAMP, strains containing eae-␥1 had Tt values (11.6 min) significantly shorter than those for strains containing eae-␤ (16.9 min), eae-ε (17 min), or eae-␪ (18.4 min) (P ⬍ 0.05). For qPCR, the vast majority of specificity tests accurately de-

April 2014 Volume 80 Number 8

tected the 156 strains, with the overall mean CT values ranging from 12.8 cycles for the eae qPCR to 20 cycles for the O103 qPCR (data not shown). However, false-negative results were consistently generated by the stx2 qPCR for two strains (ESC0603 and N5789) carrying the stx2g gene. In contrast, the CT values did not vary significantly among strains possessing different stx2 or eae subtypes (P ⬎ 0.05; data not shown). Assay sensitivity. Table 2 summarizes LAMP and qPCR sensitivity when testing 10-fold serial dilutions of STEC strains belonging to the seven adulterant serogroups. In pure culture testing, the LAMP suite consistently detected 10 to 20 CFU/reaction of target STEC strains. In one or two out of three repeats, stx2 and O45 LAMPs were capable of detecting the respective STEC strains at an even lower concentration, i.e., 1 CFU per reaction. However, the stx2 and eae LAMP assays were less sensitive (up to 100-fold) for strains carrying certain target gene subtypes, e.g., stx2c, eae-␤, and eae-␪. The detection limits for qPCR assays consistently fell between 1 and 20 CFU per reaction for all assay targets and their subtypes (Table 2). Assay sensitivity in spiked produce is also summarized in Table 2. For the uninoculated controls, aerobic plate counts averaged 104 to 105 CFU/g in lettuce and spinach varieties and 106 to 108 CFU/g in sprouts varieties, and all target genes tested negative by LAMP and qPCR (data not shown). In lettuce and spinach varieties, the LAMP suite detected down to 105 CFU per 25 g produce (approximately 103 CFU/g, equivalent to 2 CFU per reaction). In alfalfa (but not clover and mung bean) sprouts, 10-fold higher cell concentrations (106 CFU/25 g) were needed for accurate detection by the stx1, O111, and O145 LAMPs. Regardless of produce type or

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Assay target

Wang et al.

TABLE 3 Effect of six DNA extraction methods on eae LAMP and eae qPCR detection of STEC in baby spinach samples spiked with 1.2 to 1.8 CFU/ 25 g of Escherichia coli O157:H7 strain MDL 3562 (eae-␥1) and tested after cold storage and various enrichment periodsc Avg qPCR CT (no. of cycles) after enrichment for:

Avg LAMP Tt (min) after enrichment for: Method

6h

8h

10 h

24 h

24.5a 22.2a 20.6 ⫾ 1.2b

16.4 ⫾ 0.6BC 18.7 ⫾ 1.0B 14.8 ⫾ 0.7C

18.4 ⫾ 1.9AB 13.0 ⫾ 0.3C 18.2 ⫾ 0.4B 12.5 ⫾ 0.5C

14.7 ⫾ 0.2A 13.0 ⫾ 0.4B 13.5 ⫾ 0.2B 12.2 ⫾ 0.5C

21.7 ⫾ 1.9

15.6 ⫾ 1.0C 21.6 ⫾ 3.3A

13.0 ⫾ 0.3C 19.7 ⫾ 0.4A

13.0 ⫾ 0.2B 13.1 ⫾ 0.7B

6h

8h

10 h

24 h

32.5 ⫾ 0.9

29.1 ⫾ 0.8B 27.1 ⫾ 0.4C 27.9 ⫾ 0.2BC

25.0 ⫾ 0.4C 25.0 ⫾ 0.8C 30.5 ⫾ 0.1B 21.1 ⫾ 0.4D

23.6 ⫾ 0.2B 19.2 ⫾ 0.6D 21.4 ⫾ 0.6C 19.4 ⫾ 0.3D

27.1 ⫾ 1.1C 35.7 ⫾ 0.7A

20.5 ⫾ 1.3D 34.1 ⫾ 0.6A

19.6 ⫾ 0.3D 24.6 ⫾ 0.1A

33.47a

a

One out of three repeats was detected by LAMP or qPCR. Two out of three repeats were detected by LAMP. c In each column within the data for LAMP or qPCR, Tt or CT values followed by different uppercase letters are significantly different (P ⬍ 0.05). b

variety, the stx2 and eae LAMPs were less sensitive (up to 1,000fold) for strains containing stx2c, eae-␤, and eae-␪ subtypes. In comparison, all of the qPCR assays detected down to 105 CFU/25 g in lettuce, spinach, and sprouts varieties. Small variations in detection limit (10-fold) among varieties of the same produce type were observed by either LAMP or qPCR (Table 2). Effect of DNA extraction methods. Table 3 compares the effects of six DNA extraction methods on detection of E. coli O157:H7 by eae LAMP and eae qPCR in baby spinach samples spiked with 1.2 to 1.8 CFU/25 g of E. coli O157:H7 strain MDL 3562 (eae-␥1). Positive LAMP results were obtained by all six methods but at different enrichment times. For samples enriched for 6 h, the method with the PrepMan Ultra sample preparation reagent was the only one that consistently gave positive LAMP results. The methods with FTA Elute and raw enrichment broth required 8 and 10 h of enrichment, respectively, to generate positive LAMP results. For qPCR, 8 h of enrichment was needed for the majority of methods and 10 h was needed for the two-step centrifugation method. Boiling facilitated LAMP and qPCR detection, as evidenced by lower Tt and CT values in boiled samples and/or the shorter enrichment time needed for detection. Among

all six methods, the method with FTA Elute tended to generate the highest Tt or CT values at each enrichment period in baby spinach (Table 3). In other spinach varieties, the minimum enrichment time required for LAMP detection was 6 h (8 h for qPCR) when the method with two-step centrifugation followed by boiling or the PrepMan Ultra sample preparation reagent was used (data not shown). However, at least 10 h of enrichment was required for both LAMP and qPCR when using FTA Elute and at least 24 h of enrichment was required for the other three methods (raw enrichment broth, boiling preparation, and two-step centrifugation). In two lettuce varieties, regardless of the DNA extraction method, samples enriched for 6 to 8 h were accurately detected by LAMP and qPCR (data not shown). In three sprouts varieties spiked with 1.2 ⫻ 102 to 1.8 ⫻ 102 CFU/25 g of STEC cells, 6 to 8 h of enrichment was sufficient for LAMP and qPCR detection, except for alfalfa and clover sprouts by FTA Elute, which needed 10 h (data not shown). Rapid detection of low levels of STEC in spiked produce. Table 4 summarizes the LAMP and qPCR results in baby spinach samples spiked with 1.2 to 1.8 CFU/25 g of STEC strains and tested after

TABLE 4 Comparison of the LAMP suite and qPCR assays with baby spinach samples spiked with 1.2 to 1.8 CFU/25 g of STEC strains and tested after cold storage and various enrichment periodsc Assay target

Target gene and subtype

Stx1 Stx2 Stx2 Stx2 Intimin Intimin Intimin Intimin O26 O45 O103 O111 O121 O145 O157 a b c

stx1a stx2a stx2c stx2d eae-␤ eae-␥1 eae-ε eae-␪ wzy (O26) wzy (O45) wzx (O103) wzy (O111) wzy (O121) wzx (O145) wzy (O157)

Avg LAMP Tt (min) after enrichment for: Strain SJ9 SJ9 CVM 9790 CVM 9322 SJ9 CVM 9790 CVM 9322 SJ13 SJ3 SJ9 CVM 9322 SJ13 ESC0601 CVM 9790 MDL 3562

6h

8h

10 h

24.6 ⫾ 2.3 33.4 ⫾ 0.8A 34 ⫾ 0.6b 30.5 ⫾ 0.6B 27.4 ⫾ 1.1A 14.1 ⫾ 0.8B 20.6 ⫾ 0.3B 23.3 ⫾ 0.3B 22.0 ⫾ 1.1B 22.9 ⫾ 2.6B 30.6 ⫾ 2.5B 24.7 ⫾ 0.4B 21.8 ⫾ 0.8B 28.8 ⫾ 1.5B 31.9 ⫾ 3.0A A

39.3a 34.0 ⫾ 1.2A 16.7 ⫾ 0.2A 25.4 ⫾ 0.5A 25.8 ⫾ 1.4A 28.6 ⫾ 1.7A 28.4 ⫾ 2.6A 37.0 ⫾ 3.9A 28.3 ⫾ 1.2A 25.5 ⫾ 1.0A 35.5 ⫾ 2.8A

Avg qPCR CT (no. of cycles) after enrichment for: 24 h

19.9 ⫾ 1.0 28.7 ⫾ 0.6B 31.0 ⫾ 1.3A 27.9 ⫾ 0.6C 23.1 ⫾ 0.8B 12.5 ⫾ 0.3C 17.2 ⫾ 0.6C 21.8 ⫾ 0.4B 19.8 ⫾ 0.7C 20.3 ⫾ 1.7B 26.5 ⫾ 1.8B 24.2 ⫾ 1.0B 19.7 ⫾ 0.7C 24.0 ⫾ 1.2C 25.9 ⫾ 5.3B B

6h

17.1 ⫾ 0.6 26.0 ⫾ 1.0C 29.0 ⫾ 1.6A 27.0 ⫾ 0.8C 19.4 ⫾ 0.3C 12.0 ⫾ 0.3C 15.7 ⫾ 0.4D 22.0 ⫾ 1.4B 18.5 ⫾ 0.5C 18.8 ⫾ 1.4C 25.5 ⫾ 0.8C 24.2 ⫾ 1.8B 19.6 ⫾ 0.7C 22.5 ⫾ 1.9C 25.1 ⫾ 3.3B B

8h

33.9 ⫾ 0.7 34.47a

A

32.8 ⫾ 0.5A 31.9 ⫾ 0.4A 32.4 ⫾ 0.6A 29.4 ⫾ 0.5A 31.1 ⫾ 0.4A 32.3 ⫾ 0.7A 29.8 ⫾ 0.8A 32.5 ⫾ 0.5A 31.8 ⫾ 0.8A

10 h

29.8 ⫾ 0.5 30.8 ⫾ 0.4A 28.8 ⫾ 0.3A 29.1 ⫾ 0.4B 30.8 ⫾ 0.5A 27.8 ⫾ 0.6B 28.3 ⫾ 0.4B 26.7 ⫾ 0.9B 24.6 ⫾ 0.1B 30.5 ⫾ 0.7A 28.0 ⫾ 0.6B 26.7 ⫾ 1.1B 28.1 ⫾ 0.9B 27.5 ⫾ 1.9B 28.4 ⫾ 0.9A B

24 h

25.8 ⫾ 0.9 26.4 ⫾ 0.4B 23.8 ⫾ 1.0B 24.3 ⫾ 0.6C 26.6 ⫾ 0.9B 23.7 ⫾ 1.1C 23.7 ⫾ 0.8C 25.1 ⫾ 0.8C 20.6 ⫾ 0.3C 25.9 ⫾ 0.5B 23.3 ⫾ 0.4C 24.7 ⫾ 1.1C 25.5 ⫾ 0.1C 21.8 ⫾ 1.9C 20.4 ⫾ 1.9B C

19.1 ⫾ 0.9D 18.9 ⫾ 0.4C 21.1 ⫾ 0.6C 22.7 ⫾ 0.4D 19.6 ⫾ 0.4C 21.6 ⫾ 0.6D 22.0 ⫾ 0.6D 25.0 ⫾ 0.1C 17.0 ⫾ 0.8D 19.0 ⫾ 0.3C 21.9 ⫾ 0.4D 24.4 ⫾ 0.2C 24.5 ⫾ 0.7C 18.7 ⫾ 0.8D 19.2 ⫾ 1.6C

One out of three repeats was detected by LAMP or qPCR. Two out of three repeats were detected by LAMP. In each row within the data for LAMP or qPCR, Tt or CT values followed by different uppercase letters are significantly different (P ⬍ 0.05).

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Raw enrichment broth Boiling preparation Two-step centrifugation Two-step centrifugation followed by boiling PrepMan Ultra reagent FTA Elute

LAMP Assays for STEC in Produce

DISCUSSION

Upon comprehensive evaluation using 156 bacterial strains and eight produce items, the STEC LAMP suite was demonstrated to be rapid (10 to 45 min), reliable (no false-positive or false-negative results), and robust (under conditions mimicking real-world contamination events, e.g., surface contamination, cold storage). Coupled with an effective DNA extraction method, the assays accurately detected low levels (1.2 to 1.8 CFU/25 g) of STEC in lettuce and spinach varieties (but not sprouts) after 6 to 8 h of enrichment. A similar trend of detection was observed for qPCR. This is the first study comparing LAMP and qPCR for detecting STEC strains with various stx and eae subtypes in multiple types of high-risk produce using conditions close to those from real-world applications. Few studies have closely examined the ability of PCR and qPCR to detect STEC stx or eae subtypes (39–41), and to our knowledge, only one recent study explored the ability of LAMP to detect eae variants (42). There are currently 3 stx1 subtypes (stx1a, stx1c, and stx1d), 7 stx2 subtypes (a through g), and about 30 eae subtypes (␣, ␤, ␥, ε, etc.) (37, 43). The presence of eae subtypes (primarily ␤, ␥, and ␪) and certain closely related stx2 subtypes (stx2a, stx2c, and stx2d) is strongly associated with severe human illness, such as HUS (36, 43). In a recent produce survey of STEC, the stx1a and stx2a subtypes were the most common, followed by stx2d and stx2c, and only 2 to 3% of strains had the stx2e and stx2g subtypes, while the eae subtypes present were ␤, ␥, and ε (43). The LAMP suite was capable of detecting all of the major target gene subtypes evaluated in this study, consisting of two stx1 subtypes (stx1a and stx1c), four stx2 subtypes (stx2a, stx2c, stx2d, and stx2g), and four eae subtypes (␤, ␥, ε, and ␪). The ability to detect all 27 eae variants tested was also reported in a recent eae LAMP study (42). Such broad specificity is not unexpected, since both eae LAMP assays employed primers in the highly conserved N-terminal region (670 amino acids) of all intimin subtypes (36). In contrast, the eae qPCR primers were located outside this region, increasing the assay’s vulnerability for false-negative results. Due to the inability of their qPCR assays to detect some eae and stx subtypes (stx1d, stx2e, stx2f, and stx2g), the current USDA Microbiology Laboratory Guidebook 5B has adopted eae and stx primers/probes developed elsewhere,

April 2014 Volume 80 Number 8

modified the O145 probe, and included a commercially available qPCR (BAX system; Dupont Qualicon) as an alternative method (44). Other studies also reported the failure of some PCR/qPCR assays to detect genetically distant stx subtypes, e.g., stx2b, stx2f, and stx2g (40, 45). A recent evaluation of seven commercially available qPCR systems for stx subtypes also returned variable results (41). Therefore, careful selection and evaluation of primers/ probes are critical in developing these NAATs. Besides the inability of the stx2 qPCR to detect two strains containing stx2g, the specificities of LAMP and qPCR did not obviously differ for the 156 strains tested. Upon initial development, LAMP assays targeting STEC virulence genes (32) and seven O serogroups (33) were shown to be 100% specific by testing 90 and 120 strains, respectively. However, the specificity of qPCR assays evaluated in this study was not reported when initially developed (35), except that the O157 qPCR targeting the wzy gene was reported to be 100% specific (34). Such high specificity also agreed with that for other LAMP assays for STEC and E. coli O157 (20, 24, 29, 30). Interestingly, significant differences in LAMP Tt values among virulence gene-based assays or serogroup-specific assays or among strains possessing different stx2 or eae subtypes were observed in this study, suggesting variations in assay amplification efficiency. Despite this, all Tt values observed in positive LAMP assays were below 31 min, indicating robust detection (46). An entirely new set of seven STEC strains was selected to evaluate assay sensitivity. The detection limits of the LAMP suite (1 to 20 CFU per reaction in pure culture) closely mimicked those in previous studies (32, 33) and also fell within the range (0.7 to 100 CFU per reaction) reported for other STEC LAMP assays (20–24, 29–31). One important finding in the present study is that the stx2 and eae LAMPs were less sensitive (up to 100-fold) in strains containing stx2c, eae-␤, and eae-␪ subtypes, likely due to mismatches on LAMP primer sequences between these subtypes and those used as prototypes for primer design, i.e., sequences with GenBank accession numbers X07865 (stx2a) and Z11541 (eae-␥1) (32). Previously, eae LAMP showed inferior sensitivity in STEC strains 97-3250 and 3215-99 (32), which have now been confirmed by PCR-RFLP, similar to other strains used in this study, to harbor the eae-␤ and eae-␪ subtypes, respectively (data not shown). Conversely, qPCR assays were capable of detecting various stx2 and eae subtypes without compromised sensitivity, possibly due to the use of fewer pairs of primers in qPCR. Lettuce, spinach, and sprouts were tested since they have historically been involved in STEC outbreaks (10–13). The recent produce survey also identified spinach, lettuce, and cilantro (not sprouts) to be most problematic in STEC contamination (43). The seven serogroup-specific LAMP assays have been evaluated in baby spinach and romaine lettuce (33) but not virulence-based assays (32). In previous studies, inoculation usually occurred in produce homogenates rather than on the surface of intact produce, and samples were tested directly without cold storage (33). In the present study, with surface inoculation and aging treatment, the LAMP suite achieved robust and accurate detection of 105 to 106 CFU/25 g (i.e., 103 to 104 CFU/g, equivalent to 2 to 20 CFU per reaction) of STEC in all produce varieties without enrichment, which was comparable to the detection limits reported previously (33). Consistent with pure culture sensitivity data, inferior sensitivity was also observed when testing strains with certain target gene subtypes (stx2c, eae-␤, and eae-␪) in produce. The small variations (10-fold) observed among varieties of the same

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cold storage and various enrichment periods. After 6 h of enrichment, all LAMP assays in the suite successfully detected such low levels of STEC in baby spinach, except for the stx1 and O157 LAMPs and the stx2 and eae LAMPs in strains carrying stx2c and eae-␤, respectively. Significantly higher Tt values were observed for samples enriched for 6 or 8 h than those enriched for longer periods (P ⬍ 0.05). A similar trend of detection was observed for qPCR, although the O45 qPCR rather than the stx1 qPCR failed to detect STEC with the 6-h enrichment period (Table 4). In other spinach and lettuce varieties, both LAMP and qPCR achieved accurate detection after 6 to 8 h of enrichment, except for stx2 LAMP in savoy spinach spiked with CVM 9790 (stx2c), which required 10 h of enrichment (data not shown). In contrast, neither LAMP nor qPCR detected such low levels (1.2 to 1.8 CFU/25 g) of STEC strains in sprouts varieties even after 24 h of enrichment (data not shown). The 102-CFU/25 g inoculum resulted in positive LAMP and qPCR results in sprouts after 6 to 8 h of enrichment for 10 target gene and subtype combinations (stx1a, stx2a, stx2c, stx2d, eae-␤, eae-␥1, O26, O45, O103, and O145). All 15 targets/ subtypes were detected in sprouts samples spiked with 103 CFU/25 g after 6 to 8 h of enrichment (data not shown).

Wang et al.

ACKNOWLEDGMENTS We thank Baoguang Li and Narjol Gonzalez-Escalona from the U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition (FDA/CFSAN), for providing some of the STEC strains and the LA-320C real-time turbidimeter, respectively. This work was supported in part by a research grant (SCB11064) from the Center for Produce Safety at the University of California, Davis.

REFERENCES 1. Karmali MA, Gannon V, Sargeant JM. 2010. Verocytotoxin-producing Escherichia coli (VTEC). Vet. Microbiol. 140:360 –370. http://dx.doi.org /10.1016/j.vetmic.2009.04.011.

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produce type indicated the effect of produce variety on sample processing and downstream assay, underscoring the need for commodity-specific evaluation. With 6 to 8 h of enrichment, the LAMP suite detected 1.2 to 1.8 CFU/25 g of STEC in lettuce and spinach varieties, even among strains containing target gene subtypes (stx2c, eae-␤, and eae-␪) for which assay sensitivity was inferior. Similar detection capabilities using PCR or qPCR for STEC in leafy greens were obtained, usually with 24 h of enrichment (47, 48). In the present study, for samples enriched for longer than 8 h, more stable (i.e., statistically insignificant) and shorter Tt values were observed. In contrast, enrichment periods longer than 6 h were found to be disadvantageous for the recovery of STEC from salad samples with low levels of contamination (49). The temperature difference used for enrichment (37°C versus 42°C) may account for the discrepancy. In sprouts varieties, both LAMP and qPCR failed to detect this low level (1.2 to 1.8 CFU/25 g) of STEC with up to 24 h of enrichment. Previous studies in Japan applying LAMP in alfalfa and radish sprouts reported 90 to 100% detection rates in samples contaminated with 1 to 20.8 CFU/25 g of STEC after overnight enrichment (20, 25, 28). A multiplex PCR was also recently shown to detect 7 to 58 CFU/25 g of STEC in alfalfa, leek, and soy sprouts (50). It is noteworthy that sprouts samples had rather abundant natural flora, which was at levels 2 to 3 log units higher than those in leafy greens. Further studies are needed to examine whether this normal flora in sprouts or natural compounds released from sprouts during processing may affect STEC survival during enrichment. If necessary, procedures such as acid treatment or immunomagnetic separation may be incorporated to enhance STEC detection in sprouts (51). DNA extraction is a critical step in molecular-based NAATs. The PrepMan Ultra sample preparation reagent yielded the best results, but the other five methods also generated satisfactory results for LAMP and qPCR with samples enriched for up to 24 h. Previously, the PrepMan Ultra reagent has been widely used to extract STEC DNA from a wide range of food samples, including fresh produce (52). FTA Elute had the advantage of preserving sample DNAs for up to 2 years and without centrifugation steps; however, the final DNA amount extracted was approximately 100-fold less concentrated than that obtained by the other methods, thus requiring more cells in the enrichment broth or a prolonged enrichment time. In conclusion, the LAMP suite was demonstrated to be a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing, providing a valuable tool for the produce industry and regulatory agencies to better identify contamination risks and ensure produce safety, therefore reducing produce-related STEC outbreaks and illnesses.

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