DuPont qualicon BAX system real-time PCR assay for Escherichia coli O157:H7

Evaluations were conducted to test the performance of the BAX System Real-Time PCR assay, which was certified as Performance Tested Method 031002 for screening E. coli O157:H7 in ground beef, beef trim, spinach, and lettuce. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the FDA-BAM and the USDA-FSIS culture methods, but with a significantly shorter time to result. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined affect the performance of the assay. An accelerated shelf life study determined an initial 36 month shelf life for the test kit.     

Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201

The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.     

Reveal 8-Hour Test System for detection of Escherichia coli O157:H7 in raw ground beef, raw beef cubes, and iceberg lettuce rinse: collaborative study.

Five different food types were analyzed by the Reveal for E. coli O157:H7 8-Hour Test System (Reveal 8) and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method or the U.S. Department of Agriculture Food Safety Inspection Service (FSIS) culture method for the presence of E. coli O157:H7. A total of 27 laboratories representing academia and private industry in the United States and Canada participated. Food types were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. During this study, 1,110 samples and controls were analyzed by both the Reveal 8 and by BAM or FSIS by each of the collaborators (2,220 samples in total). For each set of samples, 740 were artificially inoculated with E. coli O157:H7, and 370 were uninoculated controls. The Reveal 8 detected 528 presumptive positives of which 487 were confirmed positive by the BAM culture method. In comparison, BAM and FSIS detected 489 of the 740 artificially contaminated samples as positive. In an additional in-house study performed only on chilled and frozen raw ground beef, 240 artificially inoculated samples were analyzed by both the Reveal 8 and by FSIS. The Reveal 8 detected and confirmed 104 samples as positive compared to 79 confirmed positive by FSIS.     

Comparison of the Reveal 20-hour method and the BAM culture method for the detection of Escherichia coli O157:H7 in selected foods and environmental swabs: collaborative study.

Four different food types along with environmental swabs were analyzed by the Reveal for E. coli O157:H7 test (Reveal) and the Bacteriological Analytical Manual (BAM) culture method for the presence of Escherichia coli O157:H7. Twenty-seven laboratories representing academia and private industry in the United States and Canada participated. Sample types were inoculated with E. coli O157:H7 at 2 different levels. Of the 1,095 samples and controls analyzed and confirmed, 459 were positive and 557 were negative by both methods. No statistical differences (p <0.05) were observed between the Reveal and BAM methods.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 [Visual Immunoprecipitate (VIP) for E. coli O157:H7] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of E. coli O157:H7 in Foods, AOAC Official Method 996.09, has been modified to use a simplified plastic housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 240 samples and controls. Results showed that the VIP for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.     

Sensitivity and specificity of the BAX for screening/Listeria monocytogenes assay: internal validation and independent laboratory study

In this study to certify the BAX for Screening/Listeria monocytogenes assay (DuPont Qualicon, Wilmington, DE), an internal evaluation was conducted on 16 food types that were simultaneously analyzed with the BAX system (BAX), and the ISO method for the detection of L. monocytogenes (ISO). No statistically significant difference in performance between the BAX and ISO methods was observed. Inclusivity/exclusivity testing showed that the BAX system was able to detect 97 of 97 (100%) of L. monocytogenes strains tested. None of 56 other Listeria species or non-Listeria tested gave a reproducible positive BAX result. Ruggedness testing demonstrated that performance of the assay was not affected by reasonable variability in the operating parameters. BAX was then submitted for independent laboratory validation. In this phase, BAX was compared with standard culture methods for the detection of L. monocytogenes in chicken (USDA-FSIS), crab meat (BAM), and milk (AOAC). This study validated product claims of sensitivity and specificity >98% in accordance with AOAC Performance Tested Method requirements.     

Visual immunoprecipitate assay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.09): collaborative study

AOAC Official Method 996.09, Visual Immunoprecipitate Assay (VIP) for Escherichia coli O157:H7, was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by VIP assay and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment procedure and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 15 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level, where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 396 test portions and controls by both methods, for a total of 792 test portions. Of the 396 paired test portions, 75 were positive and 230 were negative by both the VIP and culture methods. Eleven test portions were presumptively positive by VIP and could not be confirmed culturally; 32 were negative by VIP, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the VIP method. There was no statistical difference between results obtained with the VIP for EHEC 8 h method and the culture method except for cooked beef, where the VIP had significantly higher recovery for one inoculation level.     

Assurance enzyme immunoassay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.10): collaborative study

AOAC Official Method 996.10, Assurance Enzyme Immunoassay (EIA) for Escherichia coli O157:H7 (EHEC), was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by EIA and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment conditions and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 14 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 378 test portions and controls by both the 8 h EIA and the USDA/FSIS enrichment methods, for a total of 756 test portions. Of the 378 paired test portions, 75 were positive and 212 were negative by both methods. Thirteen test portions were presumptively positive by EIA and could not be confirmed culturally; 30 were negative by EIA, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the EIA method. There was no statistical difference between results obtained with the Assurance EIA for EHEC 8 h method and the culture method for raw ground beef. The Assurance EIA had a significantly higher recovery for cooked beef.     

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.     

Rapid and ultrasensitive E. coli O157:H7 quantitation by combination of ligandmagnetic nanoparticles enrichment with fluorescent nanoparticles based two-color flow cytometry

A novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay (LMNPs@FSiNPs-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles. The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody modified RuBpy-doped FSiNPs, and then stained with a nucleic acid dye SYBR Green I (SYBR-I). After dual-labeling with FSiNPs and SYBR-I, the enriched E. coli O157:H7 was determined using multiparameter FCM analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of the FSiNPs labelling method. Furthermore, the false positives caused by aggregates of FSiNPs conjugates and nonspecific binding of FSiNPs to background debris could be significantly decreased. This assay allowed the detection of E. coli O157:H7 in PB buffer at levels as low as 7 cells mL(-1). The total assay time including E. coli O157:H7 sample enrichment and detection was less than 4 h. An artificially contaminated bottled mineral water sample with a concentration of 6 cells mL(-1) can be detected by this method. It is believed that the proposed method will find wide applications in biomedical fields demanding higher sensitive bacterial identification.     

Evaluation of the assurance GDS for E. coli O157:H7 method and assurance GDS for shigatoxin genes method in selected foods: collaborative study

A multilaboratory collaborative study was conducted to compare the Assurance GDS for E. coli 0157:H7 method and the reference culture methods for the detection of E. coli 0157:H7 in orange juice, raw ground beef, and fresh lettuce. A separate companion assay, the Assurance GDS for Shigatoxin Genes method was also evaluated with the same test portions. Fifteen laboratories participated in the study. A Chi square analysis of each of the 3 food types at the high, low, and uninoculated control levels was performed. For all foods, the Assurance GDS for E. coli O157:H7 method and the Assurance GDS for Shigatoxin Genes method were equivalent to or better than the reference methods.     

An anti E. coli O157:H7 antibody-immobilized microcantilever for the detection of Escherichia coli (E. coli).

A silicon microcantilever sensor was developed for the detection of Escherichia coli O157:H7. The microcantilever was modified by anti-E. coli O157:H7 antibodies on the silicon surface of the cantilever. When the aquaria E. coli O157:H7 positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the E. coli O157:H7 antigen by the antibodies on the surface of the microcantilever. A negative control sample that does not contain E. coli O157:H7 antigen did not cause any bending of the microcantilever. The detection limit of the sensor was 1 x 10(6) cfu/mL when the assay time was < 2 h.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7...     

一种实用的基于化学发光和磁性纳米颗粒的E.coli O157:H7免疫鉴定方法

化学发光磁酶免疫已经被应用于检测病原体,但是由于针对相应病原体的抗体筛选和修饰等的步骤耗时费力,不适于对多种病原体进行筛查.制备了兔抗大肠杆菌(E.coli)O157:H7的免疫磁性纳米颗粒,富集病原菌后与鼠抗E.coli O157:H7的单克隆抗体形成双抗夹心,采用碱性磷酸酶标记的马抗鼠IgG与单抗结合,加入碱性磷酸酶的化学发光底物试剂3-(2'-螺旋金刚烷)-4-甲氧基-4-(3'-羟基)苯-1,2-二氧杂环丁烷磷酸检测化学发光.实验研究了底物缓冲液、碱性磷酸酶浓度对化学发光强度的影响,比较了NaBH4和甘氨酸对免疫磁珠剩余活性醛基的封闭效果以及本方法检测E.coli O157:H7的特异性和敏感性.结果表明,碱性磷酸酶与底物在c缓冲液中反应的化学发光强度最高,碱性磷酸酶浓度决定了化学发光的强度和持续时间,NaBH4对活性醛基的封闭效果优于甘氨酸,以D群宋内氏志贺氏菌、B群福氏志贺氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和霍乱弧菌及E.coli Top10f'为对照的比较实验显示,该检测方法具有良好的特异性,以1mL的菌液为检测体积时对E.coli O157:H7的检测灵敏度为103cell/mL,整个方法的检测时间约为3h.该方法适用于对多样本进行筛查.     

基于甘露糖功能化的水凝胶用于E.coli O157: H7的检测

利用伴刀豆球蛋白A(Con A)的多价结合能力, 结合水凝胶技术与核酸染色技术发展了一种基于甘露糖功能化的水凝胶检测大肠杆菌(E.coli)O157: H7的方法. 以过硫酸铵(APS)为催化剂, 四甲基乙二胺(TEMED)为加速剂, 用丙烯酰胺(AAm)、N,N-二甲基双丙烯酰胺和N-丙烯酰氧琥珀酰亚胺(NAS)合成水凝胶, 通过氨基化甘露糖与NAS发生交联反应, 制备了甘露糖功能化的水凝胶. 当甘露糖功能化的水凝胶加入与Con A共孵育后的菌悬液中时, 由于Con A既能与甘露糖特异性结合, 又能与E.coli O157: H7表面的O-抗原发生免疫反应而紧密连接, 使目标菌被捕获到水凝胶表面, 采用核酸染料SYBR Green Ⅰ对捕获细菌进行染色, 实现了对E.coli O157: H7的核酸标记, 最后通过活体荧光成像系统对水凝胶进行荧光成像, 从而实现对待测样品的检测. 研究结果表明, 该方法可应用于缓冲液体系和混合细菌样品中E.coli O157: H7的特异性检测, 且整个检测步骤包括样品预处理可在2 h内完成. 该方法成本低、易操作, 且具有较好的灵敏度, 可检出3.7×10¹ Cells/mL的目标细菌样品.     

Bead-Based Optical Immunoassay Using Quantum-Dot Labeling and Immunocomplex Dissociation for Detection of Escherichia coli O157:H7

An immunoassay for Escherichia coli O157:H7 using quantum-dot (QD) labeling and subsequent release of the QD labels from immunocomplexes has been developed. The assay principle is that anti-E. coli O157:H7 conjugated immunomagnetic beads are used to capture E. coli O157:H7; this is followed by the binding of QD labeled antibodies to form sandwich immunocomplexes (Bead-Cell-QD); a dissociation buffer then elutes QDs from immunocomplexes by denaturing antibody or lysing cell; the fluorescence signal of the eluted QDs is measured to quantify E. coli O157:H7. This proposed approach avoids the interference of bead autofluorescence in signal transduction and, thus, enhances the detection resolution, while keeping the fast magnetic separation and sandwich binding of two selective antibodies for high specificity.     

Escherichia coli O157:H7 detection limit of millimeter-sized PZT cantilever sensors is 700 cells/mL.

A composite self-excited millimeter-sized lead zirconate titanate (PZT) glass cantilever (2 mm x 1.8 mm; sensing area of 6 mm2) was fabricated for the detection of Escherichia coli (E. coli) O157:H7. The fundamental and second mode resonance in air was 10.95 +/- 0.05 kHz and 43.45 +/- 0.05 kHz, respectively. Affinity purified monoclonal antibody (anti-E. coli O157:H7) specific to the pathogen E. coli O157:H7 was immobilized at the cantilever glass tip, and then immersed in liquid containing the pathogen (70 to 7 x 10(7) cells/mL). The resonant frequency showed a reduction and reached a steady state shift of 0 +/- 5, 46 +/- 5, 260 +/- 5, and 1010 +/- 5 Hz corresponding to 0, 700, 7000, and 7 x 10(7) cells/mL. From the experiments conducted, the detection limit of the sensor was 700 cells/mL.     

纳米金标记抗体增强SPR检测大肠杆菌O157∶H7

将金纳米粒子(AuNPs)标记的大肠杆菌O157∶H7(E.coli O157∶H7)的多克隆抗体(PAb)作为二抗,采用氨基偶联法将PAb固定在传感器表面作为一抗,通过三明治方法用双通道表面等离子体子共振(SPR)传感器对E.coli O157∶H7进行检测,并与SPR直接法检测进行了比较.结果表明,直接法的检出限为103cfu/mL,线性范围为103～109cfu/mL;AuNPs增强三明治法的检出限为10 cfu/mL,线性范围为10～1010cfu/mL,灵敏度比直接法提高了100倍,且具有更宽的检测范围.本方法不仅检测时间短,而且具有良好的选择性和重现性.     

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC methods 996.09, Vip for EHEC and 996.10, assurance Eia EHEC with the reference culture method for the detection of Escherichia coli O157:H7 in beef

The Visual Immunoprecipitate Assay (VIP) method for the detection of enterohemorrhagic Escherichia coli O157:H7 (VIP for EHEC) and Assurance Enzyme Immunoassay (EIA) method for the detection of EHEC (EHEC EIA) are AOAC INTERNATIONAL Official Methods 996.09 and 996.10, respectively. A minor modification to the enrichment medium used in both methods has been developed. This modification, the BioControl modified EHEC medium (BioControl mEHEC) provides a more cost-effective procedure with performance equivalent to that of the cultural method for detection of E. coli O157:H7 in beef.