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At least 613 records · Page 34Linked to original sources
PubMed2007

The single-step multiplex reverse transcription- polymerase chain reaction assay for detecting H5 and H7 avian influenza A viruses.

Avian influenza (AI) A virus subtypes H5 and H7 cause severe disease in domestic poultry, including chickens and turkeys. Moreover, H5 and H7 AI A viruses can cross the species barrier from poultry to humans. In the present study, we have developed a single-step multiplex reverse transcription-polymerase chain reaction assay (RT-PCR) for detecting H5 and H7 AI A viruses. This assay was applied to the poultry isolates with the aim of establishing a surveillance method to monitor possible transmission to humans. Two subtype-specific primer sets capable of producing PCR products of 157 and 326 base pairs corresponding to AI A virus H5 and H7 subtypes, respectively, were utilized in a one-step and one-tube reaction. The single-step multiplex RT-PCR assay developed in this study was found to be specific for detecting H5 and H7 AI A viruses. No specific amplification bands were detected with total nucleic acids extracted from other influenza hemagglutinin subtypes and other viral pathogens. The sensitivity of this assay was about 10(3) RNA copies/microl. In conclusion, this novel single-step multiplex RT-PCR is a simple assay with high potential for rapid, specific and cost effective laboratory diagnosis of H5 and H7 AI A virus isolates from clinical specimens of poultry.

Animals↗
PubMedDate not supplied

Development and application of a multiplex polymerase chain reaction for avian respiratory agents.

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.

Animals↗
PubMed2003

Development of multiplex real-time RT-PCR as a diagnostic tool for avian influenza.

A multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) assay for the simultaneous detection of the H5 and H7 avian influenza hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) reporter dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro transcribed RNA templates, to have a reproducible detection limit for H7 of approximately 10(4) HA gene copies and approximately 10(4)-10(5) HA gene copies of H5. A direct comparison of H5-H7 multiplex RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR and virus isolation positive tracheal and cloacal swab samples obtained from various avian species and environmental swabs from live-bird markets in New York and New Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79 (95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either assay.

Animals↗
PubMedDate not supplied

Characterizing avian Escherichia coli isolates with multiplex polymerase chain reaction.

Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos,and the isolate of healthy birds that contained all the genes killed the most embryos amongthis group. These genes were not found among the non-E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.

Animals↗
PubMed2006

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae).

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.

Animals↗
PubMed2005

Ultrafast diagnosis of the genetic-related disorders using the combined technologies of multiplex PCR and multichannel microchip electrophoresis.

For the diagnosis of unexplained male infertility a multiplex PCR for 6 markers, which are well-known as candidate genes for studying male infertility and located on the human Y-chromosome, has been designed. The multiplex PCR products have been separated on a 12 channel microchip electrophoresis system, which can analyze different samples simultaneously. By combining the technologies of multiplex PCR with multichannel microchip electrophoresis, the number of the DNA markers that can be screened simultaneously is increased to be 72 marker (12 x 6) in a single run while the electrophoresis analysis time is reduced to be only 180 s.

Electrophoresis, Microchip↗
PubMed2000

Multiplex detection and quantitation of proteins on western blots using fluorescent probes.

The uses of multiplex detection methodologies are dramatically increasing as a means to increase sample throughput and to demonstrate quantitative differences between multiple targets in gene or protein expression analysis. In this study, we investigate the application of multiplex fluorescent detection for three proteins on the same Western blot using a laser-scanning imaging system, the Bio-Rad Molecular Imager FX. We show that independent detection and quantitation of multiple targets is achievable with little or no correction for fluorescent crosstalk by using fluorescent tags preferentially excited with different laser lines and detected at wavelengths that minimize fluorescence crosstalk. We demonstrate that the use of fluorescent detection methods can provide a tenfold greater quantifiable range but with two- to fourfold less sensitivity than chemiluminescent detection methodologies. Two examples of three-color multiplex detection using FITC-, Cy3- and Cy5-conjugated probes on Western blots are provided to demonstrate applications of this approach.

Actins↗
PubMed1996

Multiplex sets for the amplification of polymorphic short tandem repeat loci--silver stain and fluorescence detection.

Multiplex PCR amplification systems were developed using well-characterized, polymorphic short tandem repeat (STR) loci. Eight loci utilized in the multiplex amplifications included HUMCSF1PO, HUMTPOX, HUMTH01, HUMVWFA31, HUMF13A01, HUMFESFPS, HUMBFXIII and HUMLIPOL. From this list, three or four non-overlapping loci were simultaneously amplified, separated by denaturing polyacrylamide gel electrophoresis and visualized using silver stain or fluorescence detection. The multiplex PCR amplification systems offer a non-isotopic method for rapid, simple and accurate analysis of STR loci. This high-throughput method for DNA identification has immediate and valuable application in forensic analysis, paternity determination, tissue culture strain identification and bone marrow transplantation studies.

DNA↗
PubMed1997

Multiplex mutagenically separated PCR: diagnosis of beta-thalassemia and hemoglobin variants.

A rapid and simple method, termed multiplex mutagenically separated PCR (MS-PCR), was developed to detect several molecular defects in the hemoglobin gene in one PCR. This technique, in which different-size allele-specific primers were used, specifically amplified both normal and mutant alleles of the globin gene in the same reaction. Subsequent gel electrophoresis showed at least one of the two allelic products at the same locus or two of the several allelic products of different loci and provided a within-assay quality control for the exclusion of false-negative results. In our study, the four most common beta-thalassemia mutations, together with four other common hemoglobin variants in Chinese, were tested. Using multiplex MS-PCR 6 to 12 primers were added simultaneously into one reaction tube to identify one to four mutations. Not only is this multiplex MS-PCR method reliable and non-isotopic, the results can be obtained in less than one working day.

Alleles↗
PubMed1998

Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.

DNA, Complementary↗
PubMed1998

Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene.

Previously we described a PCR protocol for detecting the inversion in the factor VIII gene, which is a common cause of Hemophilia A. This PCR assay is challenging due to the size of the amplification (10-12 kb), the varying GC content (30%-80%) and the multiplex PCR products involved (four for carrier female). Efficient amplification of the four segments depends on three unusual modifications to standard long-distance PCR protocols: (i) very high concentrations of dimethyl sulfoxide, (ii) addition of deaza-dGTP and (iii) high concentration of Taq and Pwo DNA polymerases. One of the segments was amplified much more efficiently than the others under standard three-temperature cycling conditions (12 s at 94 degrees C, 30 s at 65 degrees C and 14 min at 68 degrees C). To facilitate the uniform amplification of the multiple regions, subcycling-PCR (S-PCR) was developed. In S-PCR, the combined annealing/elongation step is composed of subcycles of shuttling between a low and a high temperature, e.g., shuttling four times between 60 degrees and 65 degrees C. S-PCR produces consistent robust amplification of the various segments produced by wild-type, mutant and carrier individuals. S-PCR is a simple generalization of PCR, which generally may be advantageous in three contexts: (i) amplification of long segments in which the GC content varies within the segment, (ii) multiplex amplification of long segments and (iii) multiplex amplification of short segments in which the GC content varies among the segments.

Chromosome Inversion↗
PubMed2004

Multiplexed RT- PCR for high throughput screening applications.

To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.

Cell Count↗
PubMedDate not supplied

[Arthrogryposis multiplex congenita--a rare congenital stiff joints syndrome].

INTRODUCTION: Arthrogryposis multiplex congenita is not a disease but a term describing multiple congenital contractures. Etiological factors include neurological and primary myogenic diseases. This rare syndrome is present at birth and is characterized by reduced mobility of many joints. The contractures involve two or more joints with ankylosis. The accompanying musculature is hypoplastic, but multiple pterygia are also present. Arthrogryposis multiplex congenita is a heterogeneous group of disorders with the incidence rate of 6.2/100000 liveborn infants. The true incidence cannot be established, because many cases result in spontaneous miscarriages or stillbirth. More than 90% of cases are associated with birth defects. The cause of this syndrome is unknown. Many forms are not hereditary, though there are hereditary forms as well. CASE REPORT: This paper presents a case with arthrogryposis multiplex congenita. The pregnancy was not controlled regularly. During the pregnancy, oligohydramnion was detected. Due to contractures, labor ended is cesarean section. The child was born in the 34th week of gestation. Flexion and extension joint contractures were observed. Active and passive mobility of the afflicted joints was reduced. There was a limited motor function in the shoulder, elbow and wrist joints with a slight internal rotation of the shoulder joint and lower arm joints during pronation. The hips were subluxated; the feet were in equinovarus position and the fingers in ulnar deviation with partial syndactyly of the 4th and 5th fingers on the left hand. The infant had abnormal dermatoglyphics. The neck was short, and the 2nd and 3rd cervical vertebrae were fused. There was also a slight left-sided thoracic scoliosis. Trismus was present due to the existing ankylosis of the temporomandibular joint. The karyotype was normal. The serum creatinine phosphokinase was slightly elevated The electromyographic picture indicate non-specific signs of myopathy. DISCUSSION AND CONCLUSION: This is a case report of a "stiff joint syndrome". Due to the fact that data from the family history were unavailable, we could not establish the type of syndrome. However, heredity, growth and development at later age, as well as IQ, might significantly help in definite differentiation of this syndrome. In pregnancy, oligohydramnion should indicate more detailed ultrasonographic examination, as ankylosed joints can be detected in utero.

Arthrogryposis↗
PubMed2001

Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis.

To improve diagnosis of mastitis in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of the four major bacterial causes of bovine mastitis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. The target sequence was the 16S to 23S rRNA spacer regions. The performance of the assay was examined with 117 milk samples collected from a subclinically infected herd, and the diagnostic specificities and sensitivities of the multiplex PCR were compared with conventional culture. PCR was significantly more sensitive than culture for detection of S. aureus and S. uberis, but there were no significant differences in sensitivities between PCR and culture for the detection of S. agalactiae and S. dysgalactiae. The results suggest that this multiplex PCR assay could be used as an alternative method in routine diagnosis for rapid, sensitive, and specific simultaneous detection of S. aureus, S. agalactiae, S. dysgalactiae, and S. uberis in milk samples.

Animals↗
PubMed2003

Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species.

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.

Aminoglycosides↗
PubMed1999

A multiplex reverse transcription polymerase chain reaction method for the detection of foodborne viruses.

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, hepatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 bp, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were separated by agarose gel electrophoresis, and amplicon identity was confirmed by Southern transfer followed by DNA hybridization using nonradioactive, digoxigenin-labeled internal probes. When tested on mixed, purified virus suspensions, the multiplex method achieved detection limits of < or = 1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for all viruses. With further streamlining efforts such as single tube amplification and liquid hybridization, multiplex PCR offers advantages over cell culture methodology and monoplex PCR because it allows for rapid and cost-effective detection of several human enteric viruses in a single reaction tube.

Enterovirus↗
PubMed2000

A multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157:H7 in foods and bovine feces.

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was < or = 1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

Animals↗
PubMed2001

Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products.

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

Animals↗