Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Multiplex”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,639 records · Page 91Linked to original sources
PubMed2006

Multiplex PCR-RFLP methods for pfcrt, pfmdr1 and pfdhfr mutations in Plasmodium falciparum.

Plasmodium falciparum drug resistance is a major factor for the death toll of malaria. Resistance has been associated with specific single nucleotide polymorphisms (SNPs) in the parasite genes pfmdr1 (N86Y) and pfcrt (K76T) associated with quionoline antimalarial resistance, and pfdhfr (N51I, C59R, S108N) correlated with resistance of the antifolate combination sulfadoxine-pyrimethamine. These SNPs constitute the basis for the surveillance of drug resistance through high sensitive molecular methods in malaria endemic countries. In this work, we developed a multiplex PCR-RFLP protocols for the diagnosis of these molecular markers, leading to significant decreases in reagent costs, time, number of manipulations and hence human resources.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

A multiplex RT-PCR for detection of type A influenza virus and differentiation of avian H5, H7, and H9 hemagglutinin subtypes.

A multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) was developed and optimized for the detection of type A influenza virus; the assay simultaneously differentiates avian H5, H7 and H9 hemagglutinin subtypes. Four sets of specific oligonucleotide primers were used in this test for type A influenza virus, H5, H7 and H9 heamagglutinin subtypes. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 860 bp for H5, 634 bp for H7, 488 bp for H9 hemagglutinin subtypes, and 244 bp for type A influenza virus. The common set primers for type A influenza virus were able to amplify a 244 bp DNA band for any of the other subtypes of AIV. The mRT-PCR assay developed in this study was found to be sensitive and specific. Detection limit for PCR-amplified DNA products was 100 pg for the subtypes H5, H7, and H9 and 10 pg for type A influenza virus in all subtypes. No specific amplification bands of the same sizes (860, 634 and 488 bp) could be amplified for RNA of other influenza hemagglutinin subtypes, nor specific amplification bands of type A influenza (244 bp) for other viral or bacterial pathogens.

Animals↗
PubMed2006

Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae genes of Escherichia coli O157:H7.

A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to rfbE and eae, respectively, were targeted for reverse transcription, amplification, and real-time detection. A single-step RT-PCR kit containing a mixture of reverse transcriptases converted mRNA into cDNA, which was subsequently amplified by Taq polymerase included in the same kit. The real-time detection of amplification products was achieved by incorporating rfbE(O157)- and eae(O157:H7)-specific TaqMan probes in rRT-MPCR. The ability of two sets of primers and probes for specific detection of rfbE(O157) and eae(O157:H7) was initially verified by screening RNA of eight E. coli serotypes possessing different O antigens and eae alleles. These two sets of primers and probes were also tested in a standard real-time PCR (rPCR) using DNA prepared from several E. coli and non-E. coli strains to verify that only rfbE(O157)- and eae(O157:H7)-specific sequences were amplified and detected. The rRT-MPCR was then evaluated for detecting low-level contamination of EHEC O157:H7 in feces. When RNA prepared from bovine feces, which were artificially seeded with EHEC O157:H7 cells and cultured for five hours, was tested in rRT-MPCR as low as 1cfu/g of feces could be detected. The detection range for the two genes in fecal cultures was 5.1 x 10(-1)-5.1 x 10(4) cfu/g of feces. Thus, the described procedure could be applied to rapid detection of very low levels of EHEC O157:H7 using total RNA as a template. Since the presence of rfbE(O157)- and eae(O157:H7)-specific mRNA is dependent on replicating cells, rRT-MPCR could provide important information about the viability of EHEC O157:H7 in feces.

Adhesins, Bacterial↗
PubMed2024

Proteome-Scale Tissue Mapping Using Mass Spectrometry Based on Label-Free and Multiplexed Workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in healthy and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in a whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput. Since many of these existing challenges are associated with mass spectrometry-based protein identification and quantification, we performed a detailed benchmarking study of three protein quantification methods for spatial proteome mapping, including label-free, TMT-MS2, and TMT-MS3. Our study indicates label-free method provided the deepest coverages of ∼3500 proteins at a spatial resolution of 50 μm and the highest quantification dynamic range, while TMT-MS2 method holds great benefit in mapping throughput at >125 pixels per day. The evaluation also indicates both label-free and TMT-MS2 provides robust protein quantifications in identifying differentially abundant proteins and spatially covariable clusters. In the study of pancreatic islet microenvironment, we demonstrated deep proteome mapping not only enables the identification of protein markers specific to different cell types, but more importantly, it also reveals unknown or hidden protein patterns by spatial coexpression analysis.

Proteome↗
PubMed2005

A simple multiplex polymerase chain reaction assay for the identification of four environmentally relevant fungal contaminants.

Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time-consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. The polymerase chain reaction (PCR) has shown great promise in its ability to identify and quantify individual organisms from a mixed culture environment; however, the cost effectiveness of single organism PCR reactions is quickly becoming an issue. Our laboratory has developed a simple method to identify multiple fungal species, Stachybotrys chartarum, Aspergillus versicolor, Penicillium purpurogenum, and Cladosporium spp. by performing multiplex PCR and distinguishing the different reaction products by their mobility during agarose gel electrophoresis. The amplified genes include the beta-Tubulin gene from A. versicolor, the Tri5 gene from S. chartarum, and ribosomal sequences from both P. purpurogenum and Cladosporium spp. This method was found to be both rapid and easy to perform, while maintaining high sensitivity and specificity for characterizing isolates, even from a mixed culture.

Air Pollution, Indoor↗
PubMed2005

Multiplex PCR for screening of integrons in bacterial lysates.

Bacterial integrons are a useful PCR amplification target in epidemiological surveys of bacterial antibiotic resistance, and a variety of primers have been published. We describe multiplex PCR methodology to test for classes 1, 2 and 3 integron-associated integrases in boiled lysates of Gram-negative bacteria. We report on performance in Acinetobacter spp. (n=50), Enterobacteriaceae (n=76), Pseudomonas aeruginosa (n=15), Bacteroidesspp. (n=69), and in undifferentiated mixed cultures derived from perineal swabs (n=50) and endotracheal aspirates (n=8). This method achieved 100% sensitivity and specificity in simple lysates made from a range of bacteria, without requiring DNA extraction, and is recommended as an efficient screening tool for surveys of integron cassettes.

Acinetobacter↗
PubMed2005

Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.

A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.

Culture Media↗
PubMed2006

Direct and simultaneous identification of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium tuberculosis (MTB) by rapid multiplex nested PCR-ICT assay.

The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M. tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. The currently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application.

Chromatography↗
PubMed2006

A highly sensitive and multiplexed method for focused transcript analysis.

We describe a novel, multiplexed method for focused transcript analysis of tens to hundreds of genes. In this method TRAC (transcript analysis with aid of affinity capture) mRNA targets, a set of amplifiable detection probes of distinct sizes and biotinylated oligo(dT) capture probe are hybridized in solution. The formed sandwich hybrids are collected on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted, optionally amplified by a PCR using a universal primer pair and detected with laser-induced fluorescence and capillary electrophoresis. The probes were designed by using a computer program developed for the purpose. The TRAC method was adapted to 96-well format by utilizing an automated magnetic particle processor. Here we demonstrate a simultaneous analysis of 18 Saccharomyces cerevisiae transcripts from two experimental conditions and show a comparison with a qPCR system. The sensitivity of the method is significantly increased by the PCR amplification of the hybridized and eluted probes. Our data demonstrate a bias-free use of at least 16 cycles of PCR amplification to increase probe signal, allowing transcript analysis from 2.5 ng of the total mRNA sample. The method is fast and simple and avoids cDNA conversion. These qualifications make it a potential, new means for routine analysis and a complementing method for microarrays and high density chips.

Amino Acid Sequence↗
PubMed2006

Estimating amplification efficiency improves multiplex real-time PCR quantification of Bacillus licheniformis and Bacillus subtilis spores in animal feed.

A multiplex real-time PCR assay was developed for absolute quantification in animal feed of Bacillus subtilis CH201 and Bacillus licheniformis CH200 spores, which constitute the viable component of the microbial growth promoter, BioPlus 2B. Spores were lysed using a bead-beating protocol. DNA was extracted and purified from the lysates with the Qiagen DNeasy Plant Kit. Two standard curves for absolute quantification were made and tested. Standard curve-1 was made from feed samples spiked with BioPlus 2B, while standard curve-2 was made from serially diluted DNA extracted from BioPlus 2B powder. Feed samples supplemented with BioPlus 2B were quantified using both standard curves. The detection limit of the assay was 10(4) CFU g(-1) of feed. The amplification efficiency (Eff) of each PCR was determined using the LinRegPCR software and Eff differences between individual samples and standards were corrected for. When compared to plate counts, standard curve-1 slightly under-estimated the number of spores (mean=-2.47% of plate counts). A spore density-dependent Eff was found, and Eff for standard curve-1 could not be determined. Standard curve-2 over-estimated spore numbers when not corrected for individual Eff (mean=+5.46% of plate counts). Standard curve-2 Eff was independent (Eff(mean)=1.96) of spore density. The assay quantified the numbers of spores in feed samples very similar to plate counts (mean=+0.47% of plate counts), when standard curve-2 was used and individual Eff was accounted for.

Animal Feed↗
PubMed2004

Application of multiplex ligation-dependent probe analysis to define a small deletion encompassing PMP22 exons 4 and 5 in hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies arises as a result of defects at the chromosome 17p11.2-12 locus and in 84% of cases a 1.5 Mb deletion containing the PMP22 gene is detected by analysis that utilises polymorphic (CA)n repeat markers which flank this gene. We report the clinical and electrophysiological findings observed in a kindred with three members affected by HNPP due to a deletion containing exons 4 and 5 of the PMP22 gene. This small deletion cannot be detected using standard analysis with polymorphic (CA)n repeat markers and a definitive diagnosis was made by multiplex ligation-dependent probe analysis of PMP22 exons 1A-5. MLPA can be readily utilised as a routine diagnostic laboratory test to detect the common HNPP 1.5 Mb deletion, as well as the reciprocal 1.5 Mb insertion observed in CMT1A, but has the advantage over other diagnostic techniques of being able to define single exon deletions.

Adult↗
PubMed2006

Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by decreased levels of survival motor neuron protein (SMN). In the majority of cases, this decrease is due to absence of the SMN1 gene. Multiplex ligation-dependent probe amplification (MLPA) is a modern quantitative molecular method. Applied in SMA cases, it improves diagnostics by simultaneously identifying the number of copies of several target sequences in the SMN1 gene and in nearby genes. Using MLPA in clinical diagnostics, we have identified a previously unreported, partial deletion of SMN1 (exons 1-6) in two apparently unrelated Swedish families. This mutation would not have been detected by conventional diagnostic methods. This paper illustrates the broad clinical and genetic spectrum of SMA and includes reports of MLPA results and clinical descriptions of a patient with homozygous absence of SMN1 and only one SMN2 (prenatal onset SMA type 1), an asymptomatic woman with five SMN2 (lacking SMN1) and representative patients with SMA types 1, 2 and 3.

Adolescent↗
PubMed2006

Seasonality effects on schizophrenic births in multiplex families in a tropical island.

Many studies have found that individuals with schizophrenia have been born in winter months in disproportionately high numbers. Temperature and weather effects, such as hot summers or cold winters, have been among the suggested explanations for this seasonality effect. We studied the relationship between schizophrenia and season of birth in Puerto Rico, a tropical island with mild seasonal variation of temperature and virtually no cold periods. Our sample consisted of 132 subjects (57 with schizophrenia, 75 without) from 24 multiplex families. Schizophrenic family members were significantly more likely to be born during the winter months (21/57; 36.8%) than their unaffected relatives (16/75; 21.3%). These results suggest that extreme temperatures are not a sufficient explanation for the seasonality effect and that other factors associated with seasonality may have an effect on the later development of schizophrenia. The fact that a seasonality effect was found in a group likely to have an increased genetic loading for schizophrenia suggests that seasonality may be associated with a second, environmental "hit" in a "two-hit hypothesis" of schizophrenia.

Adult↗
PubMed2005

Detection of Salmonella in environmental water and sediment by a nested-multiplex polymerase chain reaction assay.

From 1995 to 2002, 53 serovars of Salmonella were isolated in the Seine estuary (France). The 3 serovars most frequently found were S. enterica serovar Typhimurium, S. enterica serovar Infantis and S. enterica serovar Virchow. A nested multiplex PCR (nm-PCR) assay was developed to detect the presence of Salmonella in estuarine water and sediment samples. The target gene used was the phase 1 flagellin fliC chromosomal gene, present in all Salmonella serovars. A set of 4 primers was first used to amplify an 890-bp sequence of the fliC gene, and then a second set of 3 primers was used for the nested PCR. The nmPCR method has been successfully tested for 28 serovars, 13 of which are of epidemiological significance. The detection limit of the assay, without any pre-enrichment step, was estimated at 1 CFU in deionized water, and at 4-5 CFU in the reaction mixture when tested on estuarine water seeded with a Salmonella strain. When the nmPCR was used together with the classical culture method in environmental samples, it gave additional positive results for 11.3% of the sediment samples and 20% of the water samples despite a high background of other bacteria. Overall, the results demonstrated that this molecular approach informed us about the contamination by Salmonella of estuarine water and sediment samples. Positive amplifications suggested the presence of Salmonella DNA and could thus provide information about a recent (culturable) or past (non-culturable, released DNA) contamination of environmental samples by this pathogenic bacteria.

DNA Primers↗
PubMed2006

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR.

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.

Abattoirs↗
PubMed2005

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR.

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.

Bacterial Typing Techniques↗
PubMed2005

Identification of naturally infected Lutzomyia intermedia and Lutzomyia migonei with Leishmania (Viannia) braziliensis in Rio de Janeiro (Brazil) revealed by a PCR multiplex non-isotopic hybridisation assay.

To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.

Animals↗
PubMed2005

Scaling up of PCR-based surveillance of bacterial meningitis in the African meningitis belt: indisputable benefits of multiplex PCR assay in Niger.

The absence of reliable laboratories for culture of Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, the three main causes of bacterial meningitis in Africa, hampers microbiological surveillance in these countries. To compensate for this situation in Niger, a multiplex single-tube PCR method has been implemented at a central level to test cerebrospinal fluid (CSF) samples. The overall confirmation rate for PCR (N=3791) was 40.8% compared with 16.0% for culture (N=945) (P<10(-6)). Among 850 CSF specimens tested by both methods, the overall confirmation rate was 29.4% for PCR and 16.4% for culture (P<10(-8)). PCR was also efficient for the CSF specimens stored in Trans-isolate medium. In conclusion, PCR assay is currently a key tool in Africa to improve microbiological surveillance of bacterial meningitis.

Culture Media↗