BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany. bjunge@bc-diagnostics.com
A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.
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BIOTECON Diagnostics GmbH, Hermannswerder 17, 14473, Potsdam, Germany, hdoerries@bc-diagnostics.com.
The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.
Bettina Scholtka,
Mandy Schneider,
Ralph Melcher,
Tiemo Katzenberger,
Daniela Friedrich,
Kornelia Berghof-Jäger,
Wolfgang Scheppach,
Pablo Steinberg
Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany.
BACKGROUND: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. METHODS: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243-1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. RESULTS: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. CONCLUSIONS: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80-90% of human sporadic CRC samples analyzed.
Georgia Department of Corrections, Atlanta 30334, USA. taussj00@dcor.state.ga.us
OBJECTIVE: To better understand the individual (e.g., attitudes and beliefs) and structural (e.g., laws and regulations) factors that influence and shape pharmacists' decisions about selling syringes to injection drug users (IDUs). DESIGN: Qualitative research. SETTING: Metropolitan Atlanta. PARICIPANTS: 20 practicing pharmacists who work in or near areas of high drug use in Atlanta, and nine pharmacists who are considered leaders in their profession in Georgia. INTERVENTIONS: Semistructured, in-depth interviews. MAIN OUTCOME MEASURES: Individual and structural factors that influence pharmacists' decisions about selling syringes to IDUs. RESULTS: Pharmacists reported that they use their professional discretion in making syringe sale decisions and that these decisions are influenced by individuals factors such as their personal attitudes and beliefs about the nature and causes of drug use, and by structural factors such as the Georgia Board of Pharmacy regulation stating that syringes cannot be sold if they will be used for an "unlawful purpose." CONCLUSIONS: IDUs' access to sterile syringes from pharmacies in Atlanta, would likely be increased by (1) providing practicing pharmacists with professional education programs that describe the broad professional support for IDU access to sterile syringes and why blood-borne infection prevention is a legitimate medical purpose for selling syringes and (2) removing or modifying the restrictive Board of Pharmacy regulation governing syringe sales.
Department of Medicine, University of California, San Francisco, USA.
OBJECTIVE: To determine the characteristics associated with health care and drug treatment utilization among a distinctly high-risk sub-population of injectors participating in a needle exchange program (NEP). METHODS: Between June 1998 and May 1999, study staff collected demographic and health services utilization data on participants of the Baltimore NEP. Odds ratios and logistic regression were used to identify the participant characteristics associateds with utilizing primary health care and drug treatment during the prior 3 years. RESULTS: Among 269 participants, 81% were African-American and 66% were male. Over half (56%) had not graduated from high school, 89% were unemployed, 70% did not have health insurance, and the median age was 39 years. Fifty-eight percent of the participants reported utilizing primary care (i.e., visited a physician or other health care provider) and 44% had utilized drug treatment during the prior 3 years. Primary care utilization was associated with age > or = 39 [adjusted odds ratio (AOR)= 1.82], having health insurance (AOR = 2.16), and exchanging a higher volume of syringes per NEP visit (AOR = 2.45). Recent drug treatment utilization was associated with African-American race (AOR = 0.41), unemployment (AOR = 2.72), having health insurance (AOR = 2.05), and exchanging a higher volume of syringes per NEP visit (AOR = 0.60). CONCLUSIONS: Health insurance was significantly associated with the recent utilization of both primary care and drug treatment, yet only one-third of NEP attenders were insured. Facilitating the uptake of health insurance services at NEP sites may improve the access to health care for drug users who are currently not utilizing the health care system.
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Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD, 20740, USA, Eric.Garber@fda.hhs.gov.
Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 mug/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.
Sulan Bai,
Jinyi Zhao,
Yaochuan Zhang,
Wensheng Huang,
Shi Xu,
Haodong Chen,
Liu-Min Fan,
Ying Chen,
Xing Wang Deng
Key Laboratory of Genetics and Biotechnology, College of Life Sciences, Capital Normal University, Beijing, 100048, People's Republic of China.
Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.
Manuela Avolio,
Paola Diamante,
Silvio Zamparo,
Maria Luisa Modolo,
Shamanta Grosso,
Paola Zigante,
Nilla Tosoni,
Rita De Rosa,
Paola Stano,
Alessandro Camporese
Microbiology and Virology Department, S. Maria degli Angeli Regional Hospital, via Montereale 24, 33170 Pordenone, Italy.
The rapid detection of pathogens in blood is critical for a favourable outcome of patients with suspected sepsis. Although blood culture (BC) is considered the gold standard for diagnosis of bloodstream infection (BSI), it often takes several days to detect the causative organism.In this study we compared BC with a commercially available multiplex real-time PCR assay to detect bacteria and fungi in blood samples from 144 patients admitted to the emergency room with suspected sepsis.Out of 144 blood samples examined, 91 (63%) were negatives by both methods and 53 (37%) were positive by at least one of the two methods.In 30 among all positives cases (56.6%) both methods identified the same organisms, in 13 cases (24.5%) BC identified organisms not detected by real-time PCR and in 10 cases (18.9%) SeptiFast PCR assay gave positive results whereas the BC was negative.In this study, we wished to compare SeptiFast results obtained by standard procedures, but future clinical studies are necessary to define SeptiFast PCR as support for blood culture in the early diagnosis of severe bloodstream infections.
Department of Environmental Sciences, Division of Genetics and Environmental Biotechnology, University of Parma, Viale G.P. Usberti 11/A, 43100, Parma, Italy.
We describe the development of a six-target real-time multiplex PCR assay with the SYBR(R) GreenER fluorescent dye, targeted to genes encoding for allergenic proteins commonly present in many processed food products (patent application pending). The assay was successfully trialled on reconstructed food matrices and on a range of commercial foodstuffs, and is proposed as a ready-to-use analytical tool for food manufacturers to detect the presence or confirm the absence of sequences encoding for important allergenic proteins of plant origin.
From the *UAB Epilepsy Center, Department of Neurology, University of Alabama at Birmingham; and daggerEEG Laboratory, Veterans Affairs Medical Center, Birmingham, Alabama.
PURPOSE:: Most seizure monitoring units use the Gotman algorithm or a variation on it for EEG spike detection, but the effect of various detection parameters on its accuracy has not been well established. The authors report sensitivities and false-positive rates for several different sets of detection parameters. METHODS:: Nine patients were studied. For each patient, 6 hours of EEG data were analyzed using five different sets of spike detection parameters including combinations of amplitude thresholds, state-dependent spike detection and advanced artifact rejection. Automated spike detections were compared with spikes found on visual EEG review. RESULTS:: Mean spike detection sensitivities for the different parameter sets ranged from 0.09 to 0.34. The highest sensitivity occurred with an amplitude threshold of 4, state-dependent spike detection turned on and advanced artifact rejection turned off. Mean rates of false-positives ranged from 4.2 to 48.6 per hour. The highest false-positive rate occurred with the same set of detection parameters that produced the highest sensitivity. CONCLUSIONS:: The sensitivity of spike detection with the Gotman algorithm is relatively low. The data favor using a lower amplitude threshold and not using advanced artifact rejection. The false-positive rate increases with improved sensitivity, but it is still within an acceptable range for clinical application.
University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112.
An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.
Food Research Division, Fisheries and Food Technological Institute (AZTI), Parque Tecnologico de Bizkaia, Astondo Bidea-Edificio 609, E-48160 Derio (Bizkaia), Spain.
Anisakis simplex has been recognized as an important cause of disease in humans and as a food-borne allergen source. Actually, this food-borne parasite was recently identified as an emerging food safety risk. An A. simplex -specific primer-probe system based on a real-time polymerase chain reaction (PCR) detection assay has been successfully optimized and validated with seafood samples. In addition, a DNA extraction procedure has been optimized to detect the presence of the nematode in food samples. The assay is a very reliable, specific, and sensitive methodology to detect the presence of traces of this parasite in seafood products, including highly processed samples. As a result, 13 sequences of cytochrome c oxidase II gene were obtained and scrutinized to calculate intra- and interspecific variabilities of 0 and 35-67%, respectively. Finally, an efficiency of 2.07 +/- 0.14 of the assay was calculated, and a limit of detection of 40 ppm parasite in 25 g of sample was also optimized. Actually, the presence of this parasite in several seafood products has been demonstrated, enforcing the necessity of a design for a good manufacturing practice protocol for the processing industry to minimize the presence of this parasite as a food-borne allergen source in seafood products.
Sara Longhi,
Antonella Cristofori,
Pamela Gatto,
Fabiana Cristofolini,
Maria Stella Grando,
Elena Gottardini
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Trento, Italy.
BACKGROUND: Accurate and updated information on airborne pollen in specific areas can help allergic patients. Current monitoring systems are based on a morphologic identification approach, a time-consuming method that may represent a limiting factor for sampling network enhancement. OBJECTIVE: To verify the feasibility of developing a real-time polymerase chain reaction (PCR) approach, an alternative to optical analysis, as a rapid, accurate, and automated tool for the detection and quantification of airborne allergenic pollen taxa. METHODS: The traditional cetyl trimethyl ammonium bromide-based method was modified for DNA isolation from pollen. Taxon-specific DNA sequences were identified via bioinformatics or literature searches and were PCR amplified from the matching allergenic taxa; based on the sequences of PCR products, complementary or degenerate TaqMan probes were developed. The accuracy of the quantitative real-time PCR assay was tested on 3 plant species. RESULTS: The setup of a modified DNA extraction protocol allowed us to achieve good-quality pollen DNA. Taxon-specific nuclear gene fragments were identified and sequenced. Designed primer pairs and probes identified selected pollen taxa, mostly at the required classification level. Pollen was properly identified even when collected on routine aerobiological tape. Preliminary quantification assays on pollen grains were successfully performed on test species and in mixes. CONCLUSIONS: The real-time PCR approach revealed promising results in pollen identification and quantification, even when analyzing pollen mixes. Future perspectives could concern the development of multiplex real-time PCR for the simultaneous detection of different taxa in the same reaction tube and the application of high-throughput molecular methods.
Wageningen University and Research Centre, Laboratory of Food Microbiology, P.O. Box 8129, 6700 EV Wageningen, The Netherlands; Nestec Ltd., Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland; Unilever, SEAC, Colworth Science Park, Sharnbrook, MK44 1LQ, United Kingdom.
Quantitative microbiological models predicting proliferation of microorganisms relevant for food safety and/or food stability are useful tools to limit the need for generation of biological data through challenge testing and shelf-life testing. The use of these models requires quick and reliable methods for the generation of growth data and estimation of growth parameters. Growth parameter estimation can be achieved using methods based on plate counting and methods based on measuring the optical density. This research compares the plate count method with two optical density methods, namely the two-fold dilution (2FD) method and the relative rate to detection (RRD) method. For model organism Bacillus cereus F4810/72, the plate count method and both optical density methods gave comparable estimates for key growth parameters. While values for the maximum specific growth rate (mumax) derived by the 2FD method and by the RRD method were of the same order of magnitude, some marked differences between both approaches were apparent. Whereas the 2FD method allowed deriving values for lag time (lambda) from the data, this was not possible with the RRD method. However, the RRD method gave much more data points per experiment and also gave more data points close to the growth boundary. This research shows that all three proposed methods can be used for parameter estimation, but that the choice of method depends on the objectives of the research.
From Roche Molecular Diagnostics, Pleasanton, California; Quality Systems Division, Bio-Rad Laboratories, Diagnostics Group, San Ramon, California; Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland; Quality Systems Division, Bio-Rad Laboratories, Irvine, California; Development, Asuragen, Inc., Austin Texas; and Research and Development, Abbott Molecular Inc., Des Plaines, Illinois.
The utility of quantitative molecular diagnostics for patient management depends on the ability to relate patient results to prior results or to absolute values in clinical practice guidelines. To do this, those results need to be comparable across time and methods, either by producing the same value across methods and test versions or by using reliable and stable conversions. Universally available standards and reference materials specific to quantitative molecular technologies are critical to this process but are few in number. This review describes recent history in the establishment of international standards for nucleic acid test development, organizations involved in current efforts, and future issues and initiatives.