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PubMed2001

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR.

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.

DNA, Bacterial↗
PubMed2001

A simple surgical technique for the treatment of steatocystoma multiplex.

BACKGROUND: Since steatocystoma multiplex is an uncommon disorder, its treatment has not been discussed in detail in the textbooks and surgical excision is the most commonly mentioned method. In this article, we describe a very simple surgical technique, which was developed by modifying previous reports. METHODS: A 29-year-old woman, diagnosed clinically and histologically as steatocystoma multiplex, was treated with this modified technique. We punctured the cysts under local anesthesia with a sharp-tipped cautery point and evacuated the contents by squeezing the cyst with a fine forceps. Then, the cyst wall was grasped by the forceps and the sacs were extracted through small holes. More than 50 cysts were treated. RESULTS: The treatment was well tolerated by the patient. No complications developed during or after the procedure. After 14 months follow up no recurrences were observed and the results were cosmetically excellent. CONCLUSIONS: This modified technique is very simple and time saving. Its cosmetic and long-term results are successful. We believe that it must be considered as the treatment of choice for steatocystoma multiplex.

Adult↗
PubMed2003

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems.

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.

DNA, Bacterial↗
PubMed1999

Multiplex PCR assay to aid in the identification of the highly transmissible Mycobacterium tuberculosis strain CDC1551.

SETTING: Mycobacterium tuberculosis strain CDC1551 outbreak area in Tennessee and Kentucky and selected locations in the USA. OBJECTIVE: Develop a PCR assay to distinguish the highly transmissible CDC1551 from strains which have similar 4-band IS6110 fingerprints. DESIGN: Compare the IS6110 insertion sites in CDC1551 with those in 10 isolates which have similar 4-band IS6110 fingerprints. Utilize unique characteristics of insertion sites in CDC1551 to design a multiplex PCR to identify this strain. RESULTS: A multiplex PCR was developed which targets an IS6110 insertion conserved in most IS6110 low copy number strains and a deletion within the direct repeat region adjacent to an IS6110 insertion. Of 139 isolates with similar 4-band fingerprints, the CDC1551 PCR pattern was generated by only the 14 outbreak associated isolates. Of 154 isolates with different fingerprints, only four generated the CDC1551 pattern and these could be distinguished from CDC1551 by their IS6110 fingerprint. CONCLUSIONS: The multiplex PCR used in conjunction with the IS6110 fingerprint should be a useful tool to aid in the continued surveillance of the outbreak area and follow the spread of this highly transmissible strain of M. tuberculosis.

Bacterial Typing Techniques↗
PubMed2004

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

Cations↗
PubMed2005

Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling.

Comparative proteomic approaches using isotopic labeling and MS have become increasingly popular. Conventionally quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with [13C6]lysine and [15N4]arginine; the other set was unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity, and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a 10-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated, and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG108 cells.

Amino Acid Sequence↗
PubMed2006

Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.

One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

Antibodies↗
PubMed1999

A family with autosomal dominant distal arthrogryposis multiplex congenita and brown syndrome.

Arthrogryposis multiplex congenita is a heterogeneous condition found in a number of different disorders and characterized by congenital joint contractures. We describe typical signs of congenital Brown syndrome (inability to elevate the affected eye actively or passively in full adduction) in three relatives with distal arthrogryposis multiplex congenita. We found a thickening of the superior oblique muscles in these patients with pain and increased intraocular pressure in upgaze. The pathogenesis of clinical and morphological findings is discussed. The association of Brown syndrome with distal arthrogryposis multiplex congenita has not been previously reported and provides us with an important point of reference in the understanding of both syndromes.

Adolescent↗
PubMed2005

Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies.

AIMS: To develop and evaluate multiplex PCR assays for Chlamydia trachomatis and Neisseria gonorrhoeae, using real-time and conventional PCR detection methodologies. METHODS: Two real-time multiplex PCR assays, using nuclease (TaqMan-ABI7500) and hybridisation (LightCycler) probe formats, and a third assay using conventional PCR with solid-phase hybridisation and colour detection, were developed. The porA pseudogene was targeted for N. gonorrhoeae, and the major outer membrane protein gene for C. trachomatis. A total of 145 urogenital specimens were tested in all assays, and the results were compared with the Roche Cobas Amplicor assay. RESULTS: There was little difference in clinical sensitivity and specificity, result discrimination and test cost for the three in-house assays. Our results showed that competitive inhibition of the PCR occurred in some samples that were positive for both organisms. CONCLUSIONS: These results highlight the suitability and versatility of three multiplex PCR methods for the detection of C. trachomatis and N. gonorrhoeae.

Adolescent↗
PubMedDate not supplied

Multiplex sequence-tagged site PCR for efficient screening of microdeletions in Y chromosome in infertile males with azoospermia or severe oligozoospermia.

The multiplex STS-PCR method was used to detect microdeletions in the long arm of the Y chromosome (Yq) of cytogenetically normal men. One hundred infertile men with azoospermia or oligozoospermia were screened with the multiplex PCR method using 58 STSs, which are specific to Yq for detecting microdeletions on this chromosome. Correlations between the microdeletions on Yq and phenotypes of spermatogenetic disturbance were also examined. Ten patients (10%) had microdeletions on Yq. Seven of the 60 azoospermic patients (11.7%), and 3 of the 40 oligozoospermic patients (7.5%) had microdeletions on Yq. None of the patients showed microdeletions in the AZFa region, but 2 had deletions in the AZFb region, another 2 in the AZFc region, including DAZ, and 1 had deletions in both the AZFb and in the AZFc, including RBM and DAZ. Single microdeletions were found in 4 patients, all of them in the AZFc around DAZ, and 1 patient had 2 microdeletions in the AZFb. The improved multiplex STS-PCR method efficiently detected microdeletions in 10% of azoospermic or severe oligozoospermic men who were cytogenetically normal. All of these microdeletions were presented in the AZFb and/or AZFc regions. This suggests that these regions contain candidate genes for spermatogenesis.

Adult↗
PubMed2004

Determination of verocytotoxin and eae gene loci by multiplex PCR in Escherichia coli O157:H7 isolated from human faeces in Northern Ireland: a four-year study of trends, 1997-2000.

This study aims to determine the distribution and frequency of verocytotoxin genes in human faecal clinical isolates of Escherichia coli O157 in Northern Ireland during the period 1997-2000, using a special four-target multiplex polymerase chain reaction (PCR) assay. One hundred and thirty two isolates of E. coli O157:H7 cultured during the four-year period (1997 [n=28]; 1998 [n=25]); 1999 (n=43); 2000 [n=36]), representing approximately 79% of total E. coli O157 laboratory isolations throughout N. Ireland, are examined for the presence of verocytotoxin gene loci (VT1, VT2 and eae) using a multiplex PCR assay. These isolates originate from the four Regional Area Health Boards that constitute the healthcare system in N. Ireland as follows: Eastern (53.8%; n=71), Northern (34.1%; n=45), Western (6.8%; n=9) and Southern (5.3%; n=7). Results showed that over 80% of these isolates possessed the VT2 and eae gene loci, with the remainder being predominantly VT1-, VT2- and eae-positive. None possessed the VT1 gene locus alone. Development and adoption of this simple four-target (three virulence and one control gene loci) multiplex PCR assay and subsequent recording of resulting verocytotoxin-typing data in a database, permitted local, rapid determination of carriage of known molecular virulence determinants of E. coli O157 isolates, which may aid in outbreak-related epidemiological investigations or other longitudinal studies.

Adolescent↗
PubMed2002

Improvement of multi-channel isolated bio-potential amplifiers using multiplexing.

We propose a simple isolated link circuit for a multi-channel bio-potential amplifier, by adapting the multiplexing/demultiplexing technique that is generally used in digital circuit design. The reduced number of isolation amplifiers, due to the multiplexing technique, results in an increase in the amount of isolation, as well as a reduction in manufacturing cost. The simple incorporation of a dead-zone in multiplexed timing, and a pull-down resistor in the multiplexed signal line, enables a multiple isolated bio-signal transmission over the shared isolation amplifier, with an affordable inter-channel cross talk.

Brain↗
PubMed2000

Study of HIV type 1 gag/env variability in The Gambia, using a multiplex DNA polymerase chain reaction.

A multiplex DNA PCR assay was developed for the simultaneous first-round amplification of HIV-1 gag and env fragments for the heteroduplex mobility assay (HMA). This assay was compared with the conventional amplification assay, using DNA extracted from PBMC samples from 30 HIV-1-seropositive individuals from The Gambia, who were enrolled between 1992 and 1997. From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as the mono-DNA PCR. Of the 28 Gambian isolates subtyped by gag/env HMA or by sequencing and phylogenetic analysis, the majority (19 of 28; 68%) were intersubtype recombinant. Fifteen of 28 (53%) samples were circulating recombinant form (CRF) CRF02.AG variants. Two isolates clustering with the previously documented Gambian isolate GM4 (previously described as an env GC recombinant) are classified as gag A/env J recombinants.

DNA, Viral↗
PubMed2000

Development of a one-tube multiplex reverse transcriptase-polymerase chain reaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments.

The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplification of the gag fragment was observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) tested positive. The multiplex RT-PCR in combination with gag and env HMA makes large-scale HIV-1 subtyping fast, simple, and more economical.

DNA, Viral↗
PubMed2003

Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques.

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.

Adult↗
PubMed2002

Cytochrome p450 genotyping by multiplexed real-time dna sequencing with pyrosequencing technology.

Individual differences in xenobiotic metabolism influence the therapeutic value of many drugs and are of major concern during the development of new drug candidates. A number of polymorphic cytochrome p450 enzymes account for a significant part of this variation. A better understanding of these genetic factors would be of value for drug development, as well as clinical practice. To fulfill the goal of a personalized medicine, methods for simple and accurate assessment of cytochrome p450 genes are required. We report on the development of multiplex assays for genotyping of the cytochrome p450 drug-metabolizing enzymes CYP2D6, CYP2C9, and CYP2C19 with Pyrosequencing technology. Eleven variable positions, representing 12 of the most frequent alleles, were scored: CYP2D6 alleles *2, *3, *4, *6, *7, *8, and *14, CYP2C19 alleles *2, *3, and *4, and CYP2C9 alleles *2 and *3. Four multiplex Pyrosequencing reactions per patient sample were performed to cover these positions, using either simplex or multiplex PCR for amplification of target DNA sequences. Unequivocal genotypes were obtained for all patient samples, and the results were validated by comparing with results obtained using PCR-RFLP. For positions addressed with both methods, the results were in complete agreement. Pyrosequencing technology offers a highly automated, rapid, and accurate method for identification of cytochrome p450 alleles, which is suitable for pharmacogenomic research, as well as for routine assessment of patient genotypes.

Alleles↗
PubMed1997

Dystrophin point mutation screening using a multiplexed protein truncation test.

We report here the first use of a multiplexed protein truncation test for the high throughput screening of dystrophin point mutations. We have developed a substantially more robust and efficient procedure incorporating large savings in cost which uses muscle biopsy or lymphocyte total RNA as the template. The entire dystrophin open reading frame is screened in only five overlapping fragments using a long RT-PCR strategy to amplify dystrophin cDNA in excess of 3.7 kb. These five fragments are uniquely transcribed and translated in vitro in a single multiplexed reaction containing magnesium ions to reduce nonspecific internal initiation of translation. We have used this system to analyze mutations in 11 Duchenne muscular dystrophy patients (10 unrelated) with previously uncharacterized mutations. A single truncating mutation was identified in all patients, which was confirmed at the genomic level. Multiplex PTT provides the most efficient method for point mutation screening in this large gene and has potential applications to several disease genes with a significant proportion of truncating mutations.

Amino Acid Sequence↗
PubMed2005

Detection of rearrangements in the NF2 gene using semi-quantitative multiplex fluorescent PCR.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to the development of bilateral vestibular schwannomas (sometimes associated with schwannomas at other locations), meningiomas, and ependymomas. Point mutations that inactivate the NF2 tumor suppressor gene, located in 22q12, have been found in 45-85% of NF2 patients; in addition, large genomic deletions can be found. To evaluate the presence of genomic NF2 rearrangements, we have developed a fluorescent semiquantitative multiplex PCR method. Briefly, short fragments corresponding to the 17 exons, the promoter region, and the 3' end of the NF2 gene were co-amplified by PCR using dye primers. An additional fragment, corresponding to another gene used as an internal control, was systematically amplified in each multiplex PCR. Initially, we validated the method by using monosomic 22q and trisomic 22 samples. The fluorescent multiplex PCR method was then used to analyze 21 NF2 individuals in which single-strand conformational polymorphism (SSCP) analysis and/or direct sequencing had revealed no NF2 point mutations; we were able to detect two deletions and one duplication in NF2 in 3 patients. In conclusion, the method we developed could easily be applied in detecting NF2 deletions and duplications. Discovering genomic duplications is invaluable because they are probably the most difficult molecular alterations to detect with conventional methods and, as a consequence, might be an underestimated cause of NF2.

Base Sequence↗