A multiplex real-time PCR method for the quantification of beef and pork fractions in minced meat.

One popular staple food in many lands is minced meat, traditionally prepared from beef and/or pork fractions. While beef is the more expensive of the two meat fractions, the possibility exists for manufacturers to fraudulently declare higher proportions of it. Additionally, the need exists to protect consumers who, out of medical or ethical reasons, reject specific meat fractions. In this work, we report on a quantitative triplex real-time PCR approach for the quantification of meat in minced meat products. With the method, beef and pork fractions are quantified employing primer and probe sequences that specifically recognise cow and pig components, against the backdrop of myostatin, a universal sequence commonly found in mammals and poultry species. The limit of detection of the qPCR method was 20 genome equivalents, while the measurement of uncertainty was determined at 1.83%. The method was validated on several commercially available minced meat products and performed well in terms of handling, reproducibility and robustness.

Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp.

AIMS: The aim of this study was to develop a real-time PCR test for differentiation between Shigella spp. and E. coli, in particular enteroinvasive Escherichia coli (EIEC). METHODS AND RESULTS: A duplex real-time PCR specific for the genes encoding for ß-glucuronidase (uidA) and lactose permease (lacY) was developed. Ninety-six isolates including 11 EIEC isolates of different serotypes and at least three representatives of each Shigella species were used for selectivity testing. All isolates tested were positive for the uidA gene. Additionally, all E. coli isolates were positive for the lacY gene, whereas no Shigella isolate tested harboured lacY. CONCLUSIONS: The duplex real-time PCR assay was found to be simple, rapid, reliable and specific. SIGNIFICANCE AND IMPACT OF THE STUDY: If possible at all, delineation of so-called inactive EIEC from Shigella spp. is cumbersome. Biochemical and serological methods are limited to specific pheno- and serotypes. This assay clearly simplifies the differentiation of both.

A multiplex one-step real-time RT-PCR assay for influenza surveillance.

For surveillance purposes real-time PCR assays for influenza viruses had to be adapted to the pandemic influenza A(H1N1)2009 strain. We combined published primers and probes for influenza A, influenza B and an internal amplification control with a detection system for influenza A(H1N1)2009 to set up a rapid, reliable, simple and cost-effective high-throughput multiplex one-step real-time RT-PCR. The workflow also includes automated sample preparation for high-throughput screening. The lower limit of detection of the multiplex assay was 3.5x10(2) RNA copies per PCR reaction. The diagnostic sensitivity of the multiplex assay was 87.7%, but increased to 99.4% for influenza-positive samples yielding C(t) values of less than 34 cycles in the respective diagnostic assay. High specificity was confirmed by sequencing and correct detection of 15 reference samples from two quality assurance studies. The multiplex PCR was introduced for surveillance of samples from a network of general practitioners and paediatricians in Bavaria, Germany during the influenza pandemic of 2009. Comparison with surveillance data from reported cases proved the reliability of the multiplex assay for influenza surveillance programmes.

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry as a tool for rapid diagnosis of potentially toxigenic Corynebacterium species in the laboratory management of diphtheria-associated bacteria.

The rapid identification of the potentially toxigenic Corynebacterium species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis is essential for diagnosis and treatment of diphtheria and diphtheria-like diseases. We used matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDIT-OF MS) in comparison with classical microbiological and molecular methods on 116 Corynebacterium strains. All 90 potentially toxigenic Corynebacterium strains collected by the German National Consiliary Laboratory on Diphtheria in a period of more than ten years were correctly identified by MALDI-TOF MS. We propose an algorithm for fast and reliable diagnosis of diphtheria incorporating MALDI-TOF MS, real-time tox PCR and Elek testing.

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods.

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1cfu/mL and 10cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.

Rapid detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in food, using multiplex real-time PCR.

A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

Prevalence of emetic Bacillus cereus in different ice creams in Bavaria.

In this study, 809 samples of ice cream from different sources were investigated by using cultural methods for the presence of presumptive Bacillus cereus. Isolates from culture-positive samples were examined with a real-time PCR assay targeting a region of the cereulide synthetase gene (ces) that is highly specific for emetic B. cereus strains. The samples were collected from ice cream parlors and restaurants that produced their own ice cream and from international commercial ice cream companies in different regions of Bavaria during the summer of 2008. Presumptive B. cereus was found in 508 (62.7%) ice cream samples investigated, and 24 (4.7%) of the isolates had the genetic background for cereulide toxin production. The level of emetic B. cereus in the positive samples ranged from 0.1 to 20 CFU/g of ice cream.

Single-nucleotide polymorphism in the SCCmec-orfX junction distinguishes between livestock-associated MRSA CC398 and human epidemic MRSA strains.

A number of real-time PCR assays for direct detection of methicillinresistant (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic T(m) value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.

Dissection of biochemical borderline phenotypes in carriers and genetic variants of medium-chain acyl-CoA dehyrogenase deficiency: implications for newborn screening [corrected].

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) represents a potentially fatal fatty acid beta-oxidation disorder. Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has been implemented worldwide, but is associated with unresolved questions regarding population heterogeneity, burden on healthy carriers, cut-off policies, false-positive and negative rates. In a retrospective case-control study, 333 NBS samples showing borderline acylcarnitine patterns but not reaching recall criteria were genotyped for the two most common mutations (c.985A>G/c.199C>T) and compared with genotypes and acylcarnitines of 333 controls, 68 false-positives, and 34 patients. c.985A>G was more frequently identified in the study group and false-positives compared to controls (1:4.3/1:2.3 vs. 1:42), whereas c.199C>T was found more frequently only within the false-positives (1:23). Biochemical criteria were devised to differentiate homozygous (c.985A>G), compound heterozygous (c.985A>G/c.199C>T), and heterozygous individuals. Four false-negatives were identified because our initial algorithm required an elevation of octanoylcarnitine (C(8)) and three secondary markers in the initial and follow-up sample. The new approach allowed a reduction of false-positives (by defining high cut-offs: 1.4 micromol/l for C(8); 7 for C(8)/C(12)) and false-negatives (by sequencing the ACADM gene of few suspicious samples). Our validation strategy is able to differentiate healthy carriers from patients doubling the positive predictive value (42-->88%) and to target NBS to MCADD-subsets with potentially higher risk of adverse outcome. It remains controversial, if NBS programs should aim at identifying all subsets of all diseases included. Because the natural course of milder variants cannot be assessed by observational studies, our strategy could serve as a general model for evaluation of MS/MS-based NBS.

[Risk factors for abundance of shiga toxin producing Escherichia coli in sewage water]. / Risikofaktoren der Belastung durch Shigatoxin-produzierende Escherichia coli (STEC) in Kläranlagen.

Shiga toxin producing Escherichia coli (STEC) in sewage influent into surface water are a potential source of human infections with STEC. Eight sewage treatment plants in Bavaria, Germany, were sampled at regular intervals from 2003 to 2004 in order to estimate STEC load and quantify risk factors. 95 of 378 samples (25 %) were tested positive for stx1and/or stx2 with PCR after enrichment culture. STEC elimination after treatment was 44 %. The following risk factors were analysed with logistic regression: location of sewage plant (rural vs. urban), treatment plant technology (two stage vs. three stage treatment) and sampling location (sewage input vs. sewage output). Rural plants had odds-ratios of 1,7 (95 % CI 1.03 - 2.69; p = 0.038) for a positive stx1 and/ or stx2 PCR result, sampling at sewage input of 2.1 (95 %CI 1.28 - 3.36; p = 0.003) and three stage plants of 1.51 (95 % CI 0.94 - 2.44; p = 0.087, not significant). Sampling after rain and after dry spells had no impact on STEC abundance (univariate Chi-square test = 0.01; df1; p = 0.920). Rural sewage plants had higher odds of STEC content. The influence of the sewage plant technology on the STEC load requires further clarification.

Ever heard of percutaneous transvenous selective coronary angiography? Unusual approach in a patient with patent ductus arteriosus.

Arterial access for coronary angiography is usually achieved by the use of direct arterial puncture or, less frequently, by arterial cutdown. We present the case of a 39-year-old woman in whom a patent ductus arteriosus was used to enter the arterial system for left ventriculography, aortography, and selective coronary angiography. To our knowledge, this is the 1st reported case of selective coronary angiography with use of a transvenous approach.

Pharmacokinetics of pinokalant, a new nonselective cation channel blocker in the rat.

The pharmacokinetics of 1-isoquinolineacetamide, 3,4-dihydro-6,7-dimethoxy-alpha-phenyl-N,N-bis [2-(2,3,4-trimethoxyphenyl)ethyl]-, monomethanesulfonate (pinokalant, salt form of the active entity LOE 908 BS, CAS 143482-63-7), a nonselective cation channel blocker, was studied in rats. Drug plasma levels declined rapidly in a polyphasic manner after intravenous bolus administration of 8.8 mg/kg LOE 908 BS. The disposition of LOE 908 BS was governed by a rapid elimination (clearance Cl = 47.7 ml/min/kg) and an extensive distribution into tissues (volume of distribution Vss = 7.21 l/kg). A dose-proportional increase of AUC and steady state concentration up to doses of 194 mg/kg (6 h infusion) was observed suggesting linear pharmacokinetics. The protein binding was very high with 99.4% to 99.7% bound to plasma proteins in the concentration range 0.26 to 2.6 micrograms/ml. The LOE 908 BS concentration-time profile in brain tissue after intravenous infusion (4.4 mg/kg/h over 4 h) paralleled those measured in plasma indicating a rapid but also low penetration of the blood-brain-barrier. The concentration-time profile of drug-related radioactivity after intravenous (bolus) administration of [14C]LOE 908 BS dropped also rapidly to approximately 16% within the first hour compared to the initial 2-min value. The drug exhibited a high biliary excretion (84% during 5 h) and, accordingly, faecal excretion was the main route of excretion (> 90%). The mass balance was complete after 96 h indicating no persistence of radioactivity in the animals. The relevance of these findings with respect to results obtained with LOE 908 BS in animal models for stroke and traumatic brain injury is discussed.

Quantitative distribution studies in animals: cross-validation of radioluminography versus liquid-scintillation measurement.

The results of a cross-validation of the radioluminography (RLG) and liquid scintillation counting (LSC) methods are presented. The methods for the determination of radioactivity concentrations were compared in 16 organs, after administration of (14)C-labeled substances to rats. LSC measurements of two kinds were used as reference methods for RLG: (1) quantitative determination of radioactivity after conventional dissection (interindividual comparison) and (2) quantitative determination of radioactivity in tissue punches taken from the whole-body sections after they had undergone RLG measurement (intraindividual comparison). Blood standards containing known concentrations were used for calibration. For statistical evaluation log-linear regression analysis of paired concentration values and organ-specific 95% confidence intervals of the log-transformed RLG/LSC concentration quotients were compared. For most organs, the slopes of the regression lines and the means of the concentration quotients were within the defined equivalence range of 0.80-1.25. Deviations were distinctly smaller in the intraindividual comparison. For some organs, however, it became clear that found concentrations were affected by self-absorption (RLG) and by differences in sample preparation (LSC). In conclusion, quantification with RLG is a reliable and reproducible method with comparable measurement precision and greater accuracy in respect of tissue localization, compared to LSC (dissection).

Precision of measurement of tissue concentrations by RLG.

Existing investigations about the precision of radioluminography (RLG) are restricted to descriptive analysis of the tissue samples. The aim of the present experiments was to obtain a general prospective statement about the precision that the RLG method can achieve. Several pharmaceutical companies in Europe participated in the experiments. Albino rats of various strains were dosed with various (14)C-labeled compounds. Whole-body sections were produced, and blood calibration scales were set up with standard radioactivity sources of dog or rat blood. Photostimulated luminescence was detected using Fuji imaging plate BAS-III. For each organ separately, variability was investigated on each of the levels: rat, section of rat, region within section, and residual, with the help of variance components. The producing company was seen as a fixed factor and adjusted for. A mixed linear model was fitted to the log-transformed data. The variance component (SD estimate) for the residual term gave the desired prospective statement about the achievable precision of the RLG method. Exponential back transformation from the logarithmic to the natural scale transformed the SD estimates to multiplication factors. In total, 29 organs were investigated. The RLG method was comparable in precision to the dissection/combustion method.

Molecular comparison of Mycoplasma hominis strains isolated from colonized women and women with various urogenital infections.

Twenty Mycoplasma hominis strains isolated from colonized women and women with various urogenital infections were investigated for genetic and antigenic homogeneity by different methods. Restriction fragment length polymorphism analysis demonstrated heterogeneity for all strains, with one exception. Two strains sequentially isolated from one patient showed identical patterns. Otherwise, no clonal clustering could be detected within the strains isolated from either of the diagnostic groups. In contrast, SDS-PAGE analysis and the comparison of the immunoblot pattern revealed antigenic similarities of strains isolated from patients with bacterial vaginosis, chorioamnionitis, premature rupture of membranes and preterm delivery as well as endometritis but showed obvious differences in comparison to strains isolated from colonized women.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

Malignant fibrous histiocytoma (MFH). A comparison of MFH in man and animals. A critical review.

This review gives information about localization and types of MFH in man and animals such as mouse, rat, cat, dog, opossum, cattle, horse and birds [e.g. mallard (a wild duck)]. Furthermore, this paper reports about cell culture dealing with MFH. The aim of this publication is to show that MFH originates from a primitive mesenchymal stem cell, fibroblastoid cell and fibroblasts. Histiocytes are, according to the literature in a small amount constituents of MFH and are reactive cells or without any meaning. In our own studies using rats [strain: Chbb: THOM (SPF)] the characteristic storiform or cartwheel pattern of tumour cells were evident. The cells were elongated, rich in endoplasmic reticulum and possessed no or very few lysosomes. The cells were predominantly fibroblasts and fibroblastoid cells. These cells were intermingled with giant cells. In other species mentioned above, the MFH showed very similar histological features. Our own results and findings obtained from the literature support our concept that the MFH represents a primitive phenotype or pleomorphic sarcoma which may differentiate in one or more directions. Histiocytes are not a neoplastic component.

Methods for the differentiation of microorganisms.

Advances in analytical and diagnostic assays based on novel nucleic acid analyses techniques have revolutionized the application of molecular differentiation of microorganisms. Phenotypic typing schemes are now broadly supplemented by new genotyping methods which allow a more refined and detailed differentiation of closely related microorganisms, bacterial strains, isolates and pathogens on the DNA level. Bio-, sero- and phagetyping, antibiotic susceptibility tests, immunoblotting as well as multilocus enzyme- or polyacrylamide gel electrophoresis are now supported by the analysis of plasmid or chromosomal DNA restriction profiles, ribotyping, pulsed-field gel electrophoresis and polymerase- or ligase-chain reaction-based methods or direct sequencing technique to differentiate microorganisms. Some of these molecular techniques are also used in the field of virology to analyse and differentiate closely related sub- or genotypes. Few examples for the analysis and investigation of these usually small genomes will also be given.