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PubMed2004

A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples.

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type 1 virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Adolescent↗
PubMed2004

Measurement of relative copy number of CDKN2A/ARF and CDKN2B in bladder cancer by real-time quantitative PCR and multiplex ligation-dependent probe amplification.

Many tumors have large homozygous deletions of the CDKN2A locus (encoding p14(ARF) and p16) and of CDKN2B (p15). Our aim was to determine which gene is the major target in bladder cancer. We used quantitative real-time PCR (RTQ-PCR) to determine copy number of p15, of p14(ARF) exon 1beta, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex PCR. Titration experiments showed that homozygous deletion could be detected in the presence of up to 30% normal DNA. Results for cell lines were compatible with previous cytogenetic analyses. Ten cell lines and 32 tumors (38.5%) had homozygous deletion of at least one target. Thirteen tumors (15.7%) had deletion of all three targets. Two tumors had deletion of p14(ARF) exon 1beta alone and four of p16 exon 2 alone. RTQ-PCR detected more homozygous deletions than duplex PCR. Finally we used a multiplex ligation-dependent probe amplification kit to provide independent confirmation of results. We conclude that with appropriate controls RTQ-PCR is a sensitive and robust method to detect copy number changes in tumors even in the presence of contaminating normal cell DNA.

Cell Cycle Proteins↗
PubMed2005

Molecular typing of West Nile Virus, Dengue, and St. Louis encephalitis using multiplex sequencing.

We report the development of an assay to simultaneously identify three of the clinically important flaviviruses (West Nile Virus, Dengue, and St. Louis encephalitis). This assay is based on the nucleotide sequence variations within a 266-bp region of the non-structural protein 5. Further, based on the nucleotide variations in the same region of the non-structural protein 5, four of the present Dengue serotypes were identified. To identify some of the subtypes of WNV we have developed a second assay using multiplex sequencing technology. The format of the result of this assay is an electropherogram of two genomic segments of the WNV genome: a 48-nucleotide sequence from the anchored core protein C and a 45-nucleotide sequence coding for the non-structural proteins (proteinase and putative helicase genes).

Base Sequence↗
PubMed2005

A novel semiquantitative fluorescence-based multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood.

A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.

Animals↗
PubMed2000

Multiplex genotype analysis of invasive carcinoma and accompanying proliferative lesions microdissected from breast tissue.

To understand the genetic basis of breast cancer in a comprehensive way, purported precursor lesions need to be analyzed at a large number of genetic marker loci and compared with each other and with the invasive components. However, the microscopic size of most of these lesions and the very small amount of material that can be obtained through microdissection limit the number of loci that can be included in the analysis. To address this issue, a multiplex genotyping approach has been developed. With this approach, polymorphic sequences at 28 marker loci were amplified simultaneously from the micro-dissected components in 5-microm paraffin-embedded breast tissue sections. The genotypes of the lesions were determined after resolving the amplified allelic products by denaturing gradient gel electrophoresis. Because the material isolated from each lesion in a single 5-microm section was sufficient for several 28-locus assays and several successive tissue sections with the same set of lesions may be prepared, it is possible to determine the genotype of each lesion at hundreds of genetic marker loci that may well cover the human genome. Analyzing a sufficient number of cases may yield information that could be used to understand the genetic basis of breast cancer development in a comprehensive way.

Alleles↗
PubMed2002

Multiplexed on-column protein digestion and capillary electrophoresis for high-throughput comprehensive peptide mapping.

A novel scheme based on multiplexed capillary electrophoresis (CE) has been developed for high-throughput, low-cost and comprehensive peptide mapping. Orthogonal peptide maps of the protein of interest were obtained by using multiple reaction conditions with three different enzymes (trypsin, pepsin, and chymotrypsin), and multiple separation conditions with six zone electrophoresis buffers and two micellar electrokinetic chromatography (MEKC) buffers. Fifteen nanoliters of two protein samples (beta-lactoglobulin A and beta-lactoglobulin B) were separately mixed on-column and digested independently at 37 degrees C for 10 min to produce peptides in a 20-capillary system. The resulting peptides were detected simultaneously at 214 nm by a photodiode array detector. The overall analysis time from reaction to detection was about 40 min.

Buffers↗
PubMed2003

Application of multiplexed capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection for the rapid measurement of endogenous extracellular signal-regulated protein kinase (ERK) levels in cell extracts.

Multiplexed (96-lane) capillary electrophoresis with laser-induced fluorescence (MCE-LIF) detection was used for the rapid analysis of extracellular signal-regulated protein kinase (ERK) levels from in vitro cell extracts. The levels of ERK enzyme in cell extracts were determined by monitoring the conversion of a fluorescent-labeled peptide substrate to a phosphorylated fluorescent-labeled peptide product using MCE-LIF. The incorporation of a fluorescent internal standard was found to improve the precision of the analysis. The enzyme assay conditions including substrate concentration, reaction time and enzyme linear range were rapidly optimized using the MCE-LIF approach for both direct and immunoprecipitation-based ERK assays. The levels of ERK from in vitro cell extracts stimulated with angiopoietin 1 (Ang1*) were determined using the MCE-LIF approach. The advantages of MCE-LIF for developing and applying enzyme assays, as well as the figures of merit for the direct and immunoprecipitation ERK assays, are discussed.

Cell Extracts↗
PubMed2003

Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology: a protein differential display approach.

The metastatic spread of tumor cells in malignant progression is known to be a major cause of cancer mortality. Protein glycosylation is increasingly being recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. The Multiplexed Proteomics (MP) approach is a new technology that permits quantitative, multicolor fluorescence detection of proteins in two-dimensional (2-D) gels and on Western blots. This methodology allows the parallel determination of both altered glycosylation patterns and protein expression level changes within a single 2-D gel experiment. The linear responses of the fluorescent dyes utilized allow rigorous quantitation of changes in protein expression over a broad 3-log linear dynamic range. Global analysis of changes in protein glycosylation and total protein expression is followed by dichromatic, lectin-based profiling methods for rapidly categorizing glycan branching structures. The MP approach was applied to whole tissue extracts of normal and cancerous liver, so that altered glycosylation modification patterns and protein expression levels could be determined. One prominent glycoprotein determined to be up-regulated in the tumor tissue was haptoglobin, an acute-phase response protein. The detection methodologies associated with the MP technology radically increase the information content of 2-D gel experiments. This new information greatly enhances the applicability of these experiments in addressing fundamental questions associated with proteome-wide glycosylation changes related to cancer.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit.

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.

Base Sequence↗
PubMed2002

Genetic dissection between silent and clinically diagnosed symptomatic forms of coeliac disease in multiplex families.

BACKGROUND: Coeliac disease has a large variation in clinical outcome. In addition to the classical disease with malabsorption, many individuals have a silent form, in which subject symptoms are missing but autoantibodies and mucosa lesions are identical to the symptomatic disease. AIM: To investigate whether differences in HLA DR-DQ genes explain the variation in outcome. MATERIALS AND METHODS: HLA DQ alleles were determined in 28 multiplex families with sibling pairs in which one had the symptomatic disease but the other had the silent form. RESULTS: No differences in the distribution of HLA DR-DQ haplotypes could be observed. The clinically diagnosed coeliac disease seemed to have earlier onset than silent coeliac disease. CONCLUSIONS: Results indicate that the major genetic susceptibility locus, HLA DQ, does not determine the exact clinical outcome of coeliac disease.

Alleles↗
PubMed2002

A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Alleles↗
PubMed2003

How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and multiplex-endonuclease genotyping approach.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

Animals↗
PubMed1996

Detection of pathogenic Yersinia enterocolitica using the multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was developed to detect the presence of the ail, yst, and virF genes of Yersinia enterocolitica simultaneously, quickly and accurately. The amplified fragment sizes were 356 base-pairs (bp) for the ail gene, 134 bp for the yst gene, and 231 bp for the virF gene. The specificity of the amplified products was confirmed by hybridization with digoxigenin-labelled oligonucleotide probes. Amplification was successful whether the template was derived from a single colony of bacteria, aliquots of boiled bacterial suspensions, from DNA extracted from pure or mixed cultures or from stool specimens. Amplification of the virF gene was also achieved from strains of Y. pseudotuberculosis carrying the 70 kb plasmid but not with preparations from other related Yersinia species or from other members of the family Enterobacteriaceae. The detection limit we established was 5-10 colony forming units per millilitre (cfu/ml) and 1.0 pg of DNA.

Animals↗
PubMed2001

Specific identification, grouping and differentiation of Campylobacter jejuni among thermophilic campylobacters using multiplex PCR.

Campylobacter species such as C. jejuni and C. coli are recognized as major causes of acute gastroenteritis world-wide. Although C. jejuni and C. coli are usually non-pathogenic in birds and animals, they cause enteric disease in humans and the source of infection is often the consumption of contaminated foodstuffs. In this paper we report on the development and use of a multiplex PCR of C. jejuni genomic DNA which yielded a PCR product with a unique polymorphic site that can be used to quickly and accurately identify and group C. Jejuni isolates from any source including DNA, cell culture, skin washings and faecal samples. The test is simple and sensitive and can detect purified DNA from a single bacterium 10(2) cells from crude lysates, 10(3) cells in seeded faeces and 120 cells/ml of washing of 1 cm2 skin fragments of chicken skin.

Animals↗
PubMed2004

A combined AFLP-multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements.

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.

Animals↗
PubMed2003

A novel multiplex-PCR for the rapid identification of Mycobacterium bovis in clinical isolates of both veterinary and human origin.

Bovine tuberculosis is a zoonotic disease that not only causes huge economic losses but also poses an important risk for human infection. The definitive identification of a clinical isolate relies on time-consuming, highly specialized and laborious biochemical tests. We have developed a method for the rapid and reliable identification of Mycobacterium bovis and for its simultaneous differentiation from other members of the M. tuberculosis complex. Furthermore, the technique also allowed us to distinguish M. tuberculosis complex members from other Mycobacterial species. The method comprises both a single PCR and a multiplex-PCR and can be confidently applied to samples of both veterinary and human origin.

Animals↗
PubMed2005

Detection of virulence-related genes by multiplex PCR in multidrug-resistant diarrhoeagenic Escherichia coli isolates from Kenya and Japan.

We compared serotypes, drug susceptibility and presence of virulence-related genes in diarrhoeagenic Escherichia coli isolates from children < 5 years from Kenya (n = 82) and Japan (n = 47). Multiplex PCR was used to detect genes coding for enteroaggregative adherence (aggR), heat-stable toxin (st), heat-labile toxin (It), verotoxin (vt), attaching and effacing mechanism (eaeA), enteroaggregative E. coli heat-stable enterotoxin 1 (astA) and enteroinvasive mechanism (invE). Kenyan E. coli O-serotypes were more diverse than those from Japan (29 vs. 12 serotypes) and exhibited high level multidrug resistance to World Health Organization (WHO) recommended antibiotics. Resistance rates to tetracycline, ampicillin and sulphamethoxazole-trimethoprim were 70.7, 65.9 and 68.3% respectively, but resistance to sulphamethoxazole-trimethoprim among the E. coli isolates from Japan was low (21%). Kenyan isolates harboured virulence-related genes in high frequency (82.9%) compared to those from Japan (25.5%) with aggR and astA being the most frequently detected genes. The presence of multiple virulence genes was associated with multidrug resistance and this merits further investigation.

Anti-Bacterial Agents↗
PubMed2000

High-throughput comprehensive peptide mapping of proteins by multiplexed capillary electrophoresis.

A novel multimodal method for peptide mapping of proteins by multiplexed capillary electrophoresis (CE) is presented. By combining charge to size separations in four different channels and micellar electrokinetic chromatography for hydrophobicity-based separations in two different channels in a 96-capillary array, peptide fragments of digested proteins were readily resolved and showed unique fingerprints. Each capillary spanned several diodes in a photodiode array (PDA) for absorption measurement. The 96 capillaries were monitored simultaneously at 214 nm by a single PDA element with 1,024 diodes, and analysis was completed within 45 min. This demonstrates that it is possible to rapidly screen biotechnological products as well as to efficiently optimize separation conditions in CE by a combinatorial approach.

Amino Acid Sequence↗