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At least 1,423 records · Page 79Linked to original sources
PubMed1993

Fabry disease: detection of gene rearrangements in the human alpha-galactosidase A gene by multiplex PCR amplification.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from lesions in the alpha-galactosidase A gene leading to deficient or absent activity of the lysosomal hydrolase. To facilitate the detection of rearrangements in this 14-kb gene, a method was developed for the PCR amplification of all seven exons from genomic DNA in a single multiplex reaction. The entire coding region and all the intron/exon boundaries were amplified as four products. Application of this method permitted the detection of all five partial deletions previously identified by Southern analysis. This rapid method can be used to identify gene rearrangements in affected hemizygotes and determine heterozygosity for at risk females in families with Fabry disease.

Base Sequence↗
PubMed1995

Deletion detection in the dystrophin gene by multiplex gap ligase chain reaction and immunochromatographic strip technology.

The purpose of this study is to demonstrate the value of a multiplex amplification and readout system. The validation was done using as a model system the detection of deletions in nine possible dystrophin exons: 4, 8, 12, 17, 19, 44, 45, 48, and 51. The amplification system was gap ligase chain reaction, adapted to amplify selected regions of multiple exons simultaneously. The amplified products were read out with an immunochromatographic methodology, adapted from that used in the Abbott product line commercialized under the name Test Pack Plus. In each amplification, the beta-globin gene was incorporated and served as a procedural control. The complete process takes < 3 hr from DNA sample to result. The procedure is therefore rapid and simple, as well as being potentially very cost effective. The combination of these two technologies is shown to be a useful tool for the determination of deletions in the nine exons of the dystrophin gene. The results of a 100-patient sample study showed concordance with cDNA and PCR in current use. Equivalent performance at two sites was shown.

Base Sequence↗
PubMed1995

A single-tube multiplex system for the simultaneous detection of 10 common cystic fibrosis mutations.

We describe a nonisotopic single-tube polymerase chain reaction (PCR) multiplex system that detects 10 of the more common cystic fibrosis (CF) mutations (delta F508, delta I507, V520F, G551D, G542X, R553X, R117H, 621 + 1G-->T, N1303K, A455E). The use of this method detects approximately 90% of the CF alleles in the British population. Five exons of the CF gene are amplified simultaneously by PCR, followed by an overnight triple enzyme restriction digest, and then resolved by high-resolution gel acrylamide electrophoresis.

Base Sequence↗
PubMed1995

Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing.

A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles.

Base Sequence↗
PubMed2004

Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses.

Genomic deletions and duplications play an important role in the etiology of human disease. Versatile tests are required to detect these rearrangements, both in research and diagnostic settings. Multiplex ligation-dependent probe amplification (MLPA) is such a technique, allowing the rapid and precise quantification of up to 40 sequences within a nucleic acid sample using a one-tube assay. Current MLPA probe design, however, involves time-consuming and costly steps for probe generation. To bypass these limitations we set out to use chemically synthesized oligonucleotide probes only. The inherent limitations of this approach are related to oligonucleotide length, and thus the number of probes that can be combined in one assay is also limited. This problem was tackled by designing a two-color assay, combining two sets of probes, each amplified by primers labeled with a different fluorophore. In this way we successfully combined 28 probes in a single reaction. The assay designed was used to screen for the presence of deletions and duplications in patients with hereditary multiple exostoses (HME). Screening 18 patients without detectable point mutations in the EXT1 and EXT2 genes revealed five cases with deletions of one or more exons: four in EXT1 and one in EXT2. Our results show that a two-color MLPA assay using only synthetic oligonucleotides provides an attractive alternative for probe design. The approach is especially suited for cases in which the number of patients to be tested is limited, making it financially unattractive to invest in cloning.

Chromosome Aberrations↗
PubMed2004

Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

Charcot-Marie-Tooth Disease↗
PubMed2001

High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise.

Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 microl PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5' exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping.

Alleles↗
PubMed2002

Determination of beta2-adrenergic receptor (ADRB2) haplotypes by a multiplexed polymerase chain reaction assay.

The beta2-adrenergic receptor (B2AR or ADRB2) is the target of beta2-agonist drugs used for bronchodilation in asthma and other respiratory diseases. The gene for this receptor (ADRB2) contains numerous single nucleotide polymorphisms (SNPs) some of which may be of pharmacogenetic relevance, although a consistent picture of genotype-phenotype relationships has yet to emerge. Recently, 12 distinct haplotypes of ADRB2were described along with preliminary evidence that certain haplotypes, rather than specific SNPs, determine differential response to the beta-agonist drug albuterol. In order to further evaluate the role of ADRB2haplotypes as pharmacogenetic determinants, simple and accurate methods for haplotyping clinical samples are required. To this end we have developed a multiplexed, allele-specific PCR assay that interrogates six ADRB2 SNPs in a manner that permits rapid and accurate assignment of ADRB2 haplotype pairs. This assay will facilitate studies of ADRB2haplotypes in phenotypes such as patient responses to beta2-agonists, bronchial hyper-responsiveness, and cardiovascular conditions.

Asthma↗
PubMed2005

High-resolution analysis of 16q22.1 in breast carcinoma using DNA amplifiable probes (multiplex amplifiable probe hybridization technique) and immunohistochemistry.

Loss of the chromosomal material at 16q22.1 is one of the most frequent genetic aberrations found in both lobular and low-grade nonlobular invasive carcinoma of the breast, indicating the presence of a tumour suppressor gene (TSG) at this region in these tumours. However, the TSG (s) at the 16q22.1 in the more frequent nonlobular carcinomas is still unknown. Multiplex Amplifiable Probe Hybridisation (MAPH) is a simple, accurate and a high-resolution technique that provides an alternative approach to DNA copy-number measurement. The aim of our study was to examine the most likely candidate genes at 16q22.1 using MAPH assay combined with protein expression analysis by immunohistochemistry. We identified deletion at 16q22.1 that involves some or all of these genes. We also noticed that the smallest region of deletion at 16q22.1 could be delineated to a 3 Mb region centromeric to the P-cadherin gene. Apart from the correlation between E-cadherin protein expression and its gene copy number, no correlation was detected between the expression of E2F-4, CTCF, TRF2 or P-cadherin with their gene's copy number. In the malignant tissues, no significant loss or decrease of protein expression of any gene other than E-cadherin was seen in association with any specific tumour type. No expression of VE-cadherin or Ksp-cadherin was detected in the normal and/or malignant tissues of the breast in these cases. However, there was a correlation between increased nuclear expression of E2F-4 and tumours with higher histological grade (p = 0.04) and positive lymph node disease (p = 0.02), suggesting that it may have an oncogenic rather than a tumour suppressor role. The malignant breast tissues also showed abnormal cytoplasmic cellular localisation of CTCF, compared to its expression in the normal parenchymal cells. In conclusion, we have demonstrated that MAPH is a potential technique for assessment of genomic imbalances in malignant tissues. Although our results support E-cadherin as the TSG in invasive lobular carcinoma, they argue against the candidacy of E2F-4, CTCF, TRF2, P-cadherin, Ksp-cadherin and VE-cadherin as TSGs in breast cancer.

Antigens, CD↗
PubMed2003

Analysis of RHD genes in Taiwanese RhD-negative donors by the multiplex PCR method.

The determination of the RhD phenotype is important in transfusion medicine. However, due to the complexity of D antigen expression, the routine serological method cannot differentiate all RhD variants. In addition, the induction of the anti-D antibody is still the major cause of severe hemolytic disease of the newborn (HDN). Therefore, it is important to understand RHD gene profiles. To analyze the RHD gene profiles of Taiwanese RhD-negative donors, the multiplex PCR method was applied to amplify RHD specific exons 3, 4, 5, 7, and 9. Based on the PCR results, the 156 RhD-negative donors were divided into 12 groups according to the different expression patterns of the RHD gene. These 12 groups were further divided into three categories: type I=Rh D(el) (21.8%); type II = partial D, containing some exons (9.0%); and type III = true RhD-negative (69.2%). The results indicated that 21.8% of RhD-negative donors in Taiwan were RhD(el), and 9% carried a part of the RHD gene. Six defined RhD variants were found in this study: four R(O) (Har), one D(Va), and two D(IVb). However, no true RhD-negative or RhD(el) donor with the CcdEe phenotype was found in this analysis.

Blood Grouping and Crossmatching↗
PubMed2001

Identification of human enterovirulent Escherichia coli strains by multiplex PCR.

Some strains of Escherichia coli are involved in enteric infections in both adults and children. However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E. coli, because of the lack of phenotypic differences. In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E. coli pathotypes. Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions. This method was applied to the detection of pathogenic E. coli isolated from 90 patients' stools specimens during an 18-month survey. Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E. coli strain was detected. This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens.

Adolescent↗
PubMed2003

Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay.

The clinical presentation of infections caused by the heterogeneous group of the respiratory viruses can be very similar. Thus, the implementation of virological assays that rapidly identify the most important viruses involved is of great interest. A new multiplex reverse transcription nested-polymerase chain reaction (RT-PCR) assay that is able to detect and type different respiratory viruses simultaneously is described. Primer sets were targeted to conserved regions of nucleoprotein genes of the influenza viruses, fusion protein genes of respiratory syncytial viruses (RSV), and hexon protein genes of adenoviruses. Individual influenza A, B, and C viruses, RSV (A and B), and a generic detection of the 48 serotypes of adenoviruses were identified and differentiated by the size of the PCR products. An internal amplification control was included in the reaction mixture to exclude false-negative results due to sample inhibitors and/or extraction failure. Detection levels of 0.1 and 0.01 TCID50 of influenza A and B viruses and 1-10 molecules of cloned amplified products of influenza C virus, RSV A and B, and adenovirus serotype 1 were achieved. The specificity was checked using specimens containing other respiratory viruses and no amplified products were detected in any case. A panel of 290 respiratory specimens from the 1999-2000 and 2000-2001 seasons was used to validate the assay. Accurately amplifying RNA from influenza and RSV prototype strains and DNA from all adenovirus serotypes demonstrates the use of this method for both laboratory routine diagnosis and surveillance of all these viruses.

Adenoviruses, Human↗
PubMed1992

Prevalence of high risk genital papillomaviruses in the Belgian female population determined by fast multiplex polymerase chain reaction.

Because in situ/filter hybridisation is not sensitive enough and because classical polymerase chain reaction (PCR) protocols are generally not sufficiently reproducible and specific, there is little accurate information on the prevalence of human papillomaviruses (HPV) 16, 18, and 33 infections in women without dyskaryotic changes of the cervix. In our hands, our Fast Multiplex PCR protocol has always been the most sensitive, specific, and reproducible DNA detection assay in all the microbiological and haematological applications we attempted (Vandenvelde C, Verstraete M, Van Beers D [1990]: Journal of Virological Methods 30:215-228; Vandenvelde C, Scheen R, Corazza F, Van Beers D [1991a]: Journal of Experimental and Clinical Hematology 33:293-297; Vandenvelde C, Scheen R, Van Beers D, Fondu P [1991b]: Journal of Experimental and Clinical Hematology 30:25-29). Using this new technique, cervical scrapes from 336 Belgian women attending the cervical cancer screening clinic were examined for the presence of these three high-risk genital papillomaviruses. Positive results were confirmed using another set of HPV-specific primers. Exactly one sixth of our population was found positive for one or more of these HPVs. Types 33 and 16 were significantly more prevalent than type 18. The nonparametric statistical analysis of the data suggests that some risk factors such as particular sexual habits, that are inversely related to age, must exist.

Adult↗
PubMed1993

High-risk genital papillomaviruses and degree of dysplastic changes in the cervix: a prospective study by fast multiplex polymerase chain reaction in Belgium.

The epidemiologic studies with the least selection bias do not support the hypothesis that HPV types 16 and/or 18 are strongly associated with cervical cancer. In this preliminary report, we describe our findings regarding type 16, 18, and 33 detection rates in 323 normal and 71 dysplastic or neoplastic cervical scrapes using fast multiplex PCR. This modified PCR technique has been shown to be the most sensitive, specific, and reproducible DNA detection method for large epidemiologic studies. The results indicate a high relative risk of increasingly severe cervical abnormality associated with the presence of high-risk HPV DNA. The analysis of the prevalence and age data according to CIN status by non-parametric statistic tests highlights the importance of other factors inversely correlated with age in the cervical transformation process.

Adult↗
PubMed1994

Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: a rapid multiplex polymerase chain reaction approach.

A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biopsied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E1 ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E1 ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells.

Chromosome Mapping↗
PubMed2006

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness.

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.

Cell Line↗
PubMed2003

Widely expressed transcripts for chemokine receptor CXCR1 in identified glutamatergic, gamma-aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain: a single-cell reverse transcription-multiplex polymerase chain reaction study.

Increasing evidence suggests that the chemokine interleukin (IL)-8/CXCL8 plays important roles in CNS development, neuronal survival, modulation of excitability, and neuroimmune response. Recently, we have shown that CXCL8 can acutely modulate ion channel activity in septal neurons expressing receptors CXCR1 and/or CXCR2. This was a surprising finding, insofar as CXCR1 expression had not been described for the mammalian brain. Here we investigated whether CXCR1 transcripts are present in other brain regions, whether they are expressed at the single-cell level in molecularly identified neurons and astrocytes, and how they are regulated during early postnatal development. In addition, possible cellular colocalization of CXCR1 and CXCR2 transcripts was examined. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that CXCR1 mRNAs were expressed in the septum, striatum, hippocampus, cerebellum, and cortex (temporoparietal and entorhinal) at different levels and appeared to be regulated independently from CXCR2 during development. By using RT multiplex PCR on acutely dissociated cells from these brain regions, we show that CXCR1 transcripts were expressed in 83% of 84 sampled neurons displaying cholinergic (choline acetyltransferase mRNAs), gamma-aminobutyric acidergic (glutamic acid decarboxylases 65 and 67 mRNAs), or glutamatergic (vesicular glutamate transporters 1 and 2 mRNAs) phenotypes. CXCR1 and CXCR2 transcripts were colocalized in 45% of neurons sampled and also were present in some glial fibrillary acidic protein mRNA-expressing astrocytes. This is the first study to demonstrate the widespread expression of CXCR1 transcripts in the brain and suggests that CXCR1 may have hitherto unsuspected roles in neuromodulation and inflammation.

Acetylcholine↗
PubMed2003

Multiplex PCR for the direct detection of Salmonella enterica from chicken, lamb and beef food products.

Three sets of known Salmonella enterica-specific primers were used collectively, for the first time, to evaluate the use of multiplex polymerase chain reaction (m-PCR) as a diagnostic tool to detect Salmonella enterica in naturally contaminated meat and poultry products. For this purpose a total of 300 samples representing the most frequently used fresh and frozen meat (beef and lamb) and poultry (chicken) products (whole, cut, ground, and processed) were collected from eight locations within Irbid city (Jordan). After an enrichment step, DNA was extracted directly from each food sample and amplified using six Salmonella enterica specific primers. Samples were also analyzed using conventional microbiological methods for the confirmation of Salmonella enterica presence. Out of 300 samples, 93 samples were positive by m-PCR. On the other hand, 67 samples were positive by conventional microbiological methods and only 26 samples were positive by m-PCR alone. The primer pairs, used here, proved to be highly specific for Salmonella enterica. Using m-PCR, Salmonella enterica detection could be achieved easily and the results had been confirmed effortlessly within a short period of time (24-36 hours) compared to 3-8 days for the conventional microbiological methods. Our findings emphasize the value of using a combination of specific primer pairs instead of a single primer pair in the detection process to minimize any chance for any inherent experimental or natural error.

Animals↗