An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR.

We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sub-lethally injured Salmonella cells.

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits.

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.

Quantitative real-time RT-PCR assay for PCA3.

OBJECTIVES: To develop a quantitative, internally standardized real-time RT-PCR assay for prostate cancer antigen 3 (PCA3), a non-translated gene found to be prostate-specific and highly overexpressed in cancer, and examine the role of PCA3 in peripheral blood with a small sample cohort. DESIGN AND METHODS: The RT-PCR assay for PCA3 is based on target-specific lanthanide probes. Peripheral blood from 91 prostatic cancer/disorder patients and healthy controls was assayed for PCA3 and prostate-specific antigen (PSA) expression. RESULTS: The dynamic range of the assay reaches over eight orders of magnitude and the limit of quantification is 800 copies per milliliter blood. Peripheral blood from 2/9 patients with metastasized cancers were PCA3 positive, whereas all the other samples were negative. Eight samples were PSA positive. CONCLUSIONS: The degree of PCA3 positivity in circulating cells from prostate cancer patients is low compared to that of PSA. In contrast to some previous reports, we found no PCA3 expression in healthy individuals.

A high-throughput population screening system for the estimation of genetic risk for type 1 diabetes: an application for the TEDDY (the Environmental Determinants of Diabetes in the Young) study.

BACKGROUND: In the TEDDY (The Environmental Determinants of Diabetes in the Young) study patient eligibility is based on the presence of some selected type 1 diabetes risk-associated human leukocyte antigen DR-DQ genotypes. A practical screening strategy was needed with efficient exclusion of ineligible patients at an early stage. Also, a simple, low-cost, and fast screening system was essential for the primary step of the risk assessment including thousands of samples. METHODS: A homogeneous genotyping system utilizing an asymmetric polymerase chain reaction (PCR) and subsequent hybridization of allele-specific probes was designed to be used as the first screening step. This assay was combined with methods further elucidating the genetic risk of type 1 diabetes to screen for high-risk individuals. RESULTS: The homogeneous assay platform allows the typing of hundreds of samples within one working day. The costs of the assay are minimal, and the reduction in hands-on time provides considerable improvements compared to the heterogeneous genotyping methods comprising separate PCR and hybridization steps. The primary selection criteria used in the first step proved to be efficient since the numbers of samples typed in subsequent stages were markedly reduced. CONCLUSIONS: The presented assay system provides a practical approach to the rapid screening of thousands of samples at low cost, a general starting point for large-scale screening studies.

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin.

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.

Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood.

OBJECTIVES: The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard. DESIGN AND METHODS: The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates. RESULTS: Reproducibility was best when large copy numbers (>5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold. CONCLUSIONS: We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.

Locked nucleic acid (LNA) probes in high-throughput genetic analysis: application to an assay for type 1 diabetes-related HLA-DQB1 alleles.

OBJECTIVES: In large-scale genetic screening, an assay that is reliable, fast and easy to perform, and straightforwardly adapted to new analytes is a necessity. We describe a one-step assay for analyzing HLA-DQB1 alleles which are associated with susceptibility to type 1 diabetes. DESIGN AND METHODS: The assay is based on asymmetric PCR amplification and a homogeneous hybridization method. The specificity of the probes was improved by substituting LNA (locked nucleic acid) for DNA at the critical bases. RESULTS: The functionality of the LNA containing probes was found to be superior compared to probes consisting of DNA only. The homogeneous assay gave a correct genotyping result in 100% of the cases, which included both extracted DNA samples and blood samples dried on sample collection cards. CONCLUSION: This homogeneous approach provides a simple method to define disease risk associated with HLA alleles for large-scale screening projects.

In vitro effects on polydextrose by colonic bacteria and caco-2 cell cyclooxygenase gene expression.

A 4-stage colon simulator and a cell culture-based human intestinal epithelial function model were combined to study the effects of a soluble fiber, polydextrose (PDX), on intestinal microbes and mucosal functions relevant to the risk of colon cancer. We observed sustained degradation of PDX throughout the different stages of the model. The fermentation was characterized by gradual degradation of PDX, production of short-chain fatty acids, and no increasing in putrefactive markers. We observed less marked effects in the microbial densities. When we applied colon fermentation metabolites obtained from the simulators with PDX to Caco-2 colon cancer cell line, a significant dose-dependent decreasing effect on cyclooxygenase-2 (COX-2) and an increasing effect on COX-3 expression levels were observed. PDX concentration appeared not to have effect on the expression levels of COX-1. Overexpression of COX-2 and decreased expression of COX-1 have been suggested to be characteristics of colon cancer. The exact physiological role of COX-3, an intron-retaining splice variant of COX-1, is not known, but it is suspected to play a role in transcriptional regulation of COX-1 and COX-2. In vitro modulation of COX expression by colon microbial fermentation products of polydextrose offers an interesting starting point for further studies on possible risk-decreasing effect of PDX on the development of colon cancer.

The genomics of probiotic intestinal microorganisms.

An intestinal population of beneficial commensal microorganisms helps maintain human health, and some of these bacteria have been found to significantly reduce the risk of gut-associated disease and to alleviate disease symptoms. The genomic characterization of probiotic bacteria and other commensal intestinal bacteria that is now under way will help to deepen our understanding of their beneficial effects.

Bifidobacterium Lactis sp. 420 up-regulates cyclooxygenase (Cox)-1 and down-regulates Cox-2 gene expression in a Caco-2 cell culture model.

Cyclooxygenases (Cox) -1 and -2 play important roles in gastrointestinal health; chronic overexpression of Cox-2 is associated with inflammatory and cancerous disease, whereas Cox-1 is expressed constitutively. We studied the effects of two probiotic (Bifidobacterium lactis sp. 420 and Lactobacillus acidophilus) and two control microorganisms (Escherichia coli and Salmonella enteritidis) and four microbial metabolites (acetate, butyrate, lactate and propionate) on the expression levels of the Cox isoforms in the enterocyte-like cell line Caco-2. Butyrate, which is anticarcinogenic, resulted in an 85% down-regulation of Cox-2 and a 37-fold increase in Cox-1 transcription. Propionate gave similar results (72% reduction of Cox-2, 23-fold induction of Cox-1), but lactate and acetate had no effect on Cox expression profile. Bifidobacterium sp. 420, which produces acetate and lactate but no butyrate or propionate, shared the Cox-1-increasing and Cox-2-silencing properties of butyrate and propionate, whereas L. acidophilus was similar to E. coli and S. enteritidis in having no effect on the Cox-1/Cox-2 ratio. For the first time, we therefore demonstrate evidence for a direct relationship between a probiotic bacterial strain and host Cox expression profile, suggesting that modulation of Cox expression may be an important factor in the potential anti-inflammatory and anticarcinogenic properties of some probiotics.

A homogeneous high-throughput genotyping method based on competitive hybridization.

OBJECTIVES: A reliable high-throughput assay system is necessary for the analysis of the ever-increasing numbers of single-nucleotide polymorphisms (SNP) relevant to genetic screening studies. We describe an assay suitable also for large-scale screening programs. DESIGN AND METHODS: The one-step assay is based on asymmetric PCR amplification of the target sequence and subsequent time-resolved fluorescence measurement. Asymmetric amplification results in a single-stranded PCR product that is detected in the amplification vessel with a highly sensitive, homogeneous hybridization method. RESULTS: A dual label, homogeneous high-throughput platform for nucleic acid sequence analysis was developed and validated using a C/T single-nucleotide polymorphism in the insulin gene as a model analyte and applied also to two other SNP-assays (poliovirus receptor A/G-polymorphism and CD86-gene exon 2 A/G-polymorphism). CONCLUSIONS: The described high-throughput genotyping technology is very competitive in price, simple in design and easily applied to any analyte sequence.

High-performance real-time quantitative RT-PCR using lanthanide probes and a dual-temperature hybridization assay.

We report a time-resolved fluorescence-based, homogeneous approach for multiplex, real-time or end-point detection of PCR products. Signal generation consists of PCR associated digestion of a 5'-labeled oligonucleotide probe, rapid cooling of the reaction mixture, and hybridization of undigested probe oligonucleotides with a complementary, shorter probe that incorporates a quencher at its 3' end. The signal coming from intact fluorescent probe molecules is, thus, quenched. The fluorophores we have used are environmentally sensitive lanthanide chelates. Their signals can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. Signal-to-noise ratios are further enhanced by the environmental sensitivity of these chelates; they exhibit a higher fluorescence intensity when free in solution than when coupled to intact probe molecules. Because of the minimal background fluorescence, the signal-to-noise ratios are higher and threshold cycles are lower than those obtained using conventional TaqMan probes. The multiplexing capacity of the assay chemistry is demonstrated through simultaneous amplification and detection of prostate specific antigen (PSA) cDNA and an internal standard mRNA (mmPSA) using probes labeled with terbium and europium. The applicability of the assay chemistry to routine clinical diagnostics is demonstrated through absolute quantification of PSA mRNA in peripheral blood.

Simultaneous quantification of prostate-specific antigen and human glandular kallikrein 2 mRNA in blood samples from patients with prostate cancer and benign disease.

BACKGROUND: Detection or quantification of circulating cancer cells has been proposed as an aid in detection and monitoring of several solid tumors. We investigated the classification accuracy of prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) mRNA copy numbers in blood for the differentiation of patients with prostate cancer (PC) and benign disease. METHODS: PSA and hK2 mRNA expression was studied in blood samples from 51 men with PC and 19 men with benign disease. Among the PC patients, 10 had organ-confined disease (pT1-pT2). We used a multiplexed reverse transcription-PCR assay with two highly target-like mRNA internal standards for the simultaneous quantification of PSA and hK2 mRNA. An external calibration curve covered the range of 10(2)-10(6) mRNA copies. RESULTS: PSA and hK2 mRNA were detected in 41 of 51 (median, 1200 copies/0.5 mL of blood) and 43 of 51 (median, 3800 copies/0.5 mL of blood) patients with PC, respectively, whereas only 1 of 19 men with benign disease was positive for both mRNAs (1500 PSA and 3100 hK2 mRNA copies/0.5 mL of blood; P <0.0001, Mann-Whitney U-test). Of the 10 patients with organ-confined PC, only 3 with low Gleason scores (< or =5) were negative for both PSA and hK2 (P = 0.02, Mann-Whitney U-test). CONCLUSIONS: The presence of PC cells in the blood circulation is an early event in PC progression, and quantitative assays for PSA and hK2 mRNA discriminate benign from PC cases. Further studies are needed to determine the diagnostic accuracy and prognostic value of the assays.