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. %; 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 = .41), unemployment (AOR = 2.72), having health insurance (AOR = 2.05), and exchanging a higher volume of syringes per NEP visit (AOR = .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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Background: The purpose of this study was to test the effectiveness of a DNA repair protocol in improving genetic testing in compromised samples, frequently encountered in Forensic Medicine.Methods: In order to stretch the experiment conditions to the limits, as far as quality of samples and DNA is concerned, we tried the repair protocol on ten ancient human teeth obtained from an equal number of skeletons from a burial site in Lerna, Middle Helladic Greece (2100 - 1700 BC). For these samples, sex was previously determined morphologically, serving as a reference to compare our molecular data with. The samples were analysed using the DNA amelogenin sex test assay prior and after DNA polymerase repair. For every individual, two molecular sex determinations were obtained by visualising PCR products on an agarose gel.Results: DNA repair enabled genetic testing in these samples. Successful amplification of the amelogenin gene was obtained only from the repaired DNA in eight out of ten samples. Prior to the repair treatment, none of these samples yielded any PCR products, thus attesting to the authenticity of the amplified sequence. The concordance between morphological and molecular analysis was in reasonable agreement (71%).Conclusions: These results reveal the impact of the repair process in studying single copy genes from low quality DNA. This protocol could facilitate molecular analysis in compromised samples, encountered in forensic medicine, as well as enable genetic studies in ancient remnants.
Tereza C Cardoso,
Deriane E Gomes,
Heitor F Ferrari,
Camila Silva-Frade,
Ana C G Rosa,
Alexandre L Andrade,
Maria Cecília R Luvizotto
UNESP- São Paulo State University, Laboratório de Virologia e Patologia Animal, Curso de Medicina Veterinária, Campus de Araçatuba, São Paulo, Brazil.
An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample (n=20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3-3 diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.
U.S. Environmental Protection Agency, Office of Pesticide Programs, Microbiology Laboratory, Environmental Science Center, Fort Meade, MD 20755-5350, USA. tomasino.stephen@epa.gov
The AOAC Use-Dilution methods, 955.14 (Salmonella enterica), 955.15 (Staphylococcus aureus), and 964.02 (Pseudomonas aeruginosa), are used to measure the efficacy of disinfectants on hard inanimate surfaces. The methods do not provide procedures to assess log density of the test microbe on inoculated penicylinders (carrier counts). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this method deficiency. Based on carrier count data collected by four laboratories over an 8 year period, a minimum log density value is proposed to qualify the test results. Carrier count data were collected concurrently with 242 Use-Dilution tests. The tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 1452 carriers. All 242 mean log densities were at least 6. (geometric mean of 1. x 10(6) CFU/carrier). The mean log densities did not exceed 7.5 (geometric mean of 3.2 x 10(7) CFU/carrier). For all microbes and OSL treatments, the mean log density (+/- SEM) was 6.7 (+/- .07) per carrier (a geometric mean of 5.39 x 10(6) CFU/carrier). The mean log density for six carriers per test showed good repeatability ( .29) and reproducibility ( .32). A minimum mean log density of 6. is proposed as a validity requirement for S. aureus and P. aeruginosa. The minimum level provides for the potential inherent variability that may be experienced by a wide range of laboratories and the slight effect due to the addition of an OSL. A follow-up report is planned to present data to support the carrier count procedure and carrier counts for S. enterica.
Frantisek Stumr,
Dana Gabrovská,
Jana Rysová,
Petr Hanák,
Jan Plicka,
Kveta Tomková,
Petr Cuhra,
Martin Kubík,
Sona Barsová,
Lenka Karsulínoví,
Hana Bulawová,
Josef Brychta
SEDIUM RD, s.r.o., Zeleznicního pluku 1361, 530 02 Pardubice, Czech Republic. stumr@nem.pce.cz
An interlaboratory study was performed in six laboratories to prove the validation of the ELISA method developed for quantitative determination of beta-lactoglobulin (BLG) in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. In-house validation of the kit did not produce false-positive results or cross-reactivity in a broad range of food matrixes containing no milk proteins. All participants obtained the BLG kit with a standard operational procedure, the list of the samples, samples, and a protocol for recording test results. The study included 14 food samples (extruded breakfast cereals, bread, two soy desserts, butter, chicken ham, chicken meat, wheat flour, long grain rice, jelly, two whey drinks, crackers, and bitter chocolate) and six spiked samples (two rice, two wheat flour, and two chicken meat). Nine samples of food matrixes containing no milk proteins showed BLG content lower than the first standard ( .15 mg/kg). Two samples of food matrixes with no milk proteins revealed BLG content higher than standard 3 (1.5 mg/100 g) and standard 4 (5. mg/100 g). Three food samples containing milk were tested as positive, and all spiked samples were evaluated as positive. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ ( .22 mg BLG/kg) and LOD ( .07 mg BLG/kg), for the kit were calculated.
Nestlé Research Center, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland. eric.poitevin@rdls.nestle.com
A single-laboratory validation (SLV) and a ring trial (RT) were undertaken to determine nine nutritional elements in food products by inductively coupled plasma-atomic emission spectroscopy in order to improve and update AOAC Official Method 984.27. The improvements involved optimized microwave digestion, selected analytical lines, internal standardization, and ion buffering. Simultaneous determination of nine elements (calcium, copper, iron, potassium, magnesium, manganese, sodium, phosphorus, and zinc) was made in food products. Sample digestion was performed through wet digestion of food samples by microwave technology with either closed or open vessel systems. Validation was performed to characterize the method for selectivity, sensitivity, linearity, accuracy, precision, recovery, ruggedness, and uncertainty. The robustness and efficiency of this method was proved through a successful internal RT using experienced food industry laboratories. Performance characteristics are reported for 13 certified and in-house reference materials, populating the AOAC triangle food sectors, which fulfilled AOAC criteria and recommendations for accuracy (trueness, recovery, and z-scores) and precision (repeatability and reproducibility RSD and HorRat values) regarding SLV and RT. This multielemental method is cost-efficient, time-saving, accurate, and fit-for-purpose according to ISO 17025 Norm and AOAC acceptability criteria, and is proposed as an improved version of AOAC Official Method 984.27 for fortified food products, including infant formula.
Shanghai Jiao Tong University, GMO Detection Laboratory, SJTU-Bor Luh Food Safety Center, School of Life Science and Biotechnology, 800 Dongchuan Rd, Shanghai 200240, People's Republic of China.
Reference molecules, as positive controls and calibrators, have been recently developed in genetically modified organism analysis as a potential substitute for reference materials derived from plant raw materials. In this study, a novel reference molecule p59122, including the revealed 5' integration sequence of maize Herculex RW (59122), was constructed that was suitable for simplex and duplex event-specific qualitative and quantitative PCR detections. The LOD values were 10 copies both for simplex and duplex qualitative PCR when p59122 was used as the calibrator. These values were comparable to those of using genomic DNA samples with .01 and .05%, approximately 5 and 25 hyploid genomic DNA copies, respectively. The absolute LOD and LOQ values were confirmed to be as low as 10 and 25 copies of p59122 DNA both in simplex and duplex quantitative systems. Furthermore, ideal quantification data with low bias, SD and RSD values were obtained from the practical samples analyses in simplex and duplex real-time PCR systems using the reference molecule p59122 as a calibrator. All these results suggested that the developed reference molecule p59122 and the qualitative and quantitative PCR detection methods are suitable for identification and quantification of GM maize 59122 and its derived products.
BBL CHROMagar Staph aureus (CSA) medium was evaluated internally and externally for the isolation and enumeration of Staphylococcus aureus in cooked roast beef, smoked salmon, and shell eggs. All food matrixes were processed according to the AOAC Official Method 975.55 and ISO 6888-1:1999. Bacterial counts of S. aureus were compared on CSA to the reference media, Baird-Parker, at low, medium, and high contamination levels. Colony counts were converted to log10 for statistical analysis. Based on the paired t-test and one-way analysis of variance, no statistical difference was noted with the CSA method compared to the AOAC Official Method for the recovery of S. aureus for all food types and contamination levels. Compared to the ISO reference method, no statistical difference was found with the CSA method for any food type or contamination level, with the exception of low-level smoked salmon. A statistical difference was seen in the internal testing with the low-level contaminated smoked salmon where CSA recovered more colonies. The external testing showed no statistical difference with smoked salmon at the low level. The correlation coefficients ranged from 92.6 to 99.4%, demonstrating good correlation for overall levels in all food types and methods. The sensitivity and specificity of the CSA method using known isolates was 100%. The results of this study demonstrate that CSA is an effective medium for the isolation, enumeration, and presumptive identification of S. aureus in cooked roast beef, smoked salmon, and shell eggs in 24 h using ISO and AOAC official methods.
BioControl Systems, Inc., 12822 SE 32nd St, Bellevue, WA 98005, USA. ptf@biocontrolsys.com
The Visual Immunoprecipitate (VIP) for the Detection of Salmonella in Foods, AOAC Official Method 999.09, has been modified to change the color of the test and control lines of 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, there were valid results from 155 samples and controls. Results showed that the modified VIP Gold for Salmonella is equivalent to the reference culture methods for the detection of Salmonella.
BioControl Systems, Inc., 12822 SE 32nd St, Bellevue, WA 98005, USA. ptf@biocontrolsys.com
The Visual Immunoprecipitate (VIP) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, has been modified to change the color of the test and control lines of the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 100 samples and controls. Results showed that the modified VIP Gold for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.
Susan Alles,
Nabina Shrestha,
Amanda Ellsworth,
Alicia Rider,
Debra Foti,
Jake Knickerbocker,
Mark Mozola
The Soleris yeast and mold method, a growth-based test system with an optical detection end point, was evaluated for its ability to detect yeast and mold contamination in a wide variety of foods. The Soleris test was used in a semiquantitative manner, in which the test result is positive or negative at a threshold level determined by the dilution and volume of sample homogenate added to the Soleris test vial. By testing at two or more threshold levels, the contamination level can be estimated. The LOD of the Soleris method is 10 CFU/g when 1 mL of a 1:10 sample homogenate is added to the test vial. In these studies, the Soleris results were compared to plate counts obtained using the U.S Food and Drug Administration/Bacteriological Analytical Manual direct plating method, and agreement between the methods was calculated. Considering results from both internal and independent laboratory trials, overall agreement between the methods was 90%. Chi-square analysis showed, with few exceptions, that results of the Soleris and direct plating methods were not statistically different. Ruggedness testing was performed, and the Soleris method was found to be robust when challenged with marginally suboptimal assay conditions. Results of inclusivity testing showed that the Soleris test vial medium supports the growth of a wide variety of yeasts and molds common to foods. Results of exclusivity testing showed that bacteria do not produce positive results, even when present in the vial in relatively high initial concentrations. The Soleris method produces results in 72 h or less and thus offers considerable time savings in comparison to other commonly used yeast and mold methods.