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PubMed2002

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases.

We used multiplex PCR follwed by sequencing to screen for mutations in the 14 exons of the RPE65 gene in early-childhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber's congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the visual cycle and mutations identified in the gene could have implications for vitamin A-based therapeutic intervention. We were able to identify a homozygous mutation (AAT --> AAG) in exon 9 in an arRP patient and a heterozygous missense transversion (AAT --> AAG) also in exon 9 of an LCA patient. We also identified a polymorphism in exon 10 (GAG --> GAA) in an arRP as well as an LCA patient. Mutation screening would be greatly facilitated by multiplex PCR which could cut down costs, labour and time involved. The nucleotide changes observed in this study could be de novo. Though a larger study has been undertaken, from the preliminary results it appears that in India the RPE65 gene seems to be less involved in causation of LCA.

Carrier Proteins↗

PubMed2000

Establishment of a multiplex PCR system to diagnose tuberculosis and other bacterial infections.

In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly conserved regions and Tb species-specific variable region of bacterial 16s rDNA. A 360 bp fragment was detected in all bacteria tested, and a 210 bp fragment was found only in Tb. 19 species of known bacteria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candida albicans and human diploid cell served as controls. It was found that both 210 bp and 360 bp fragments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of general bacteria. Candida albicans and human cells were negative for both 360 bp and 210 bp fragments. The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.

Amidohydrolases↗

PubMed2005

Multiplex PCR for detection of three exfoliative toxin serotype genes in Staphylococcus aureus.

Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control.

Adult↗

PubMed2005

Application of multiplex ARMS and SSCP/HD analysis in molecular diagnosis of cystic fibrosis in Indian patients.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The most severe, DeltaF508, mutation accounts for nearly 70% of CF cases worldwide. Besides DeltaF508, there are other point mutations, namely G542X, G551D, R553X, N1303K, and 621+1(G-->T), which are common among Caucasians. Additionally, a polyT polymorphism in intron 8 is also involved in the pathogenesis of CF. However, neither the prevalence nor the types of mutations causing CF in India are known. In this study, we aimed at estimating the frequency of the above common mutations and polymorphism in clinically suspected CF cases. We also carried out partial analysis of the CFTR gene, limited to exons 10 and 11, to identify other variations in these exons. METHODS: The multiplex amplification refractory mutation system (ARMS) test was applied for rapid simultaneous analysis of six most common CF mutations, in 100 normal and 39 elevated sweat chloride cases. The scanning of exons 10 and 11 was carried out by single-stranded conformation polymorphism/heteroduplex (SSCP/HD) analysis, followed by DNA sequencing in 50 normal and 37 elevated sweat chloride cases. A single ARMS-polymerase chain reaction assay was used to distinguish the 5T, 7T, and 9T alleles in 100 normal and 33 elevated sweat chloride cases. RESULTS: The multiplex ARMS analysis identified the DeltaF508 mutation at an allele frequency of 24% in Indian CF cases. However, the other predominant CF mutations were found to be absent. The 7T polyT variant was observed to be the most common allele, followed by the 9T, and 5T, which was the lowest. The DeltaF508 mutation was observed in all instances with the 9T variant. The SSCP/HD and DNA sequencing additionally revealed a known polymorphism (M470V, exon 10) and a known mutation [1525-1(G-->A), intron 9]. The 1525-1(G-->A) mutation, observed in a single 4-year-old male, is predicted to code for a class II defective CFTR protein. CONCLUSION: The findings of this study suggest a difference in relative frequencies and spectrum of CFTR mutations in Indian CF cases. A larger screening study of the entire CFTR gene in clinically well defined CF cases is required to delineate common mutations in the CFTR gene and enable molecular diagnosis of CF in India.

Amino Acid Substitution↗

PubMed2004

Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity.

Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays.

Animals↗

PubMed2004

Development of a sensitivity enhanced multiplexed fluorescence covalent microbead immunosorbent assay (FCMIA) for the measurement of glyphosate, atrazine and metolachlor mercapturate in water and urine.

Body burdens from exposures to pesticides may be estimated from urinary analyses of pesticide parent/metabolite concentrations. Pesticide applicators and others are often exposed to numerous unrelated pesticides, either sequentially or simultaneously. Classically, body burdens of pesticides are analyzed using chemical/instrumental analysis (CIM) or enzyme immunoassays (EIAs). Both of these technologies can usually be used to quantitate one analyte (or closely related groups of analytes) per analysis. Alternatively, multiple analytes can be measured simultaneously using a multiplexed fluorescence covalent microbead immunoassay (FCMIA). We developed a multiplexed FCMIA to simultaneously measure glyphosate (Gly), atrazine (Atz), and metolachlor mercapturate (MM) in water and urine. The assay had least detectable doses (LDDs) in water/diluted urine of 0.11/0.09 ng/ml (Gly, water/urine LDD), 0.10/0.07 ng/ml (Atz) and 0.09/0.03 ng/ml (MM). The sensitivity for the measurement of Gly was enhanced by derivatization. All assays gave linear responses from the LDDs for each respective pesticide to 300 ng/ml. There was no cross-reactivity between the three analytes. Using a 96-well microplate and an autosampler, as many as 288 separate analyses can be completed in approximately 120 min with precision, sensitivity, and specificity equivalent to, if not better, than that found when these same analytes are measured by CIM or EIA.

Acetanilides↗

PubMed1998

Carbohydrate-deficient glycoprotein syndrome type I: a new cause of dysostosis multiplex.

We report on a 1-year-old boy, with carbohydrate-deficient glycoprotein (CDG) syndrome type I due to phosphomannomutase deficiency. Radiologic examination of the skeleton revealed previously unreported bone abnormalities that could be included in a dysostosis multiplex: wide ribs, squared iliac wings, horizontal acetabular roofs, widening and modeling abnormalities of ischial and pubic bones, dorsolumbar kyphosis, and slight hook-like dysplasia of the first lumbar vertebrae. Wormian bones were also present. We suggest that these features may be due to hypoglycosylation of bone proteins and that CDG syndrome type I should be included in the differential diagnosis of dysostosis multiplex.

Congenital Disorders of Glycosylation↗

PubMed2005

Detection of human beta defensin-1 and -2 by RT-competitive multiplex PCR.

Although, the human epithelium is constantly challenged by a broad spectrum of microorganisms, invasive infections are rather rare. Recent findings suggest the expression of antimicrobial peptides by skin cells in order to provide an innate defensive barrier. In particular, peptides of the beta-defensin family offer antimicrobial activity against different pathogens including bacteria and fungi. Within this peptide family, hBD-1 is rather constitutively expressed while hBD-2 and hBD-3 expression depends on environmental conditions. The present paper introduces RT-competitive multiplex PCR as a precise tool to detect hBD-1 and hBD-2 expression on the transcriptional level. The method makes use of co-amplification of synthetic competitors along with referring wildtype targets. Competitor- and wildtype-derived products differ in size allowing signal discrimination using agarose gel electrophoresis. Regulation of gene transcripts is evaluated by comparison of competitor and corresponding wildtype signals. It was found that primary human keratinocytes stimulated with Escherichia coli cells for 8 h offered an upregulation of hBD-2 to about 2,000 fold, while hBD-1 was only marginally regulated. RT-competitive multiplex PCR is a simple and accurate method that enables new insights into defensin regulation under physiological and pathophysiological conditions.

DNA Primers↗

PubMed2003

Validation of the multiplex kit genRESMPX-2 for forensic casework analysis.

Validation studies were carried out using the commercially available PCR multiplex system genRESMPX-2. In addition to amelogenin, this system comprises the complete set of eight STR systems which are components of the German DNA database established in 1998 by the Federal Criminal Office of Germany (BKA). The minimum amount of template DNA which gave a complete DNA pattern ranged between 100 pg and 200 pg. Mixed samples could clearly be assigned from ratios between 1:5 (ACTBP2) and 1:20 (VWA, FGA). Experimental investigations with different forensic materials, environmental studies, reproducibility and precision data as well as practical casework analysis revealed that the genRESMPX-2 kit can be regarded as a sensitive, reliable and robust multiplex system even in the case of samples containing limited amounts or degraded DNA.

Animals↗

PubMed2005

Short amplicon STR multiplex for stain typing.

We developed a short tandem repeat (STR) typing kit based on DNA database systems that are included in, for example, the Interpol Standard Set of Loci recommendations (i.e., TH01, VWA, D3S1358, FGA) and the gender typing system Amelogenin. Two different multiplex sets were tested using the fluorescent dyes FAM, JOE, and VIC. The PCR results were compared to the commercially available AmpFISTR Blue kit, which contains the STRs D3S1358, VWA, and FGA. The advantage of our multiplex compared with the Blue kit was the generation of shorter amplicons (<200 bp) and the higher combined power of discrimination.

Amelogenin↗

PubMed2001

Multiplex PCR amplification of eight STR loci in Austrian and Croatian Caucasian populations.

Austrian and Croatian Caucasian population data were generated for eight tetrameric STR loci amplified in a single multiplex reaction. Fluorescent detection was employed using the ABI Prism 310 Genetic Analyzer, the 377 DNA Sequencer (ABI) and the 373A DNA Sequencer (ABI). The loci analyzed were HUMvWFA31 (vWA), HUMTH01, HUMTPOX, HUMCSF1PO, D5S818, D13S317, D7S820 and D16S539 as part of the GenePrint PowerPlex multiplex system.

Alleles↗

PubMed2005

Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for beta-thalassemia mutations.

Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HBB alleles in the Taiwanese population, using parallel minisequencing and multiplex assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was optimized for sensitivity and resolution by "mass tuning" the PinPoint assay for eight HBB SNPs. Because of the close proximity and clustering of mutations in HBB, primer extension reactions were conducted in parallel. Efficient sequential desalting using POROS and cationic exchange chromatography allowed for an unambiguous multiplex genotyping by MALDI-TOF MS. The embellishing SNP assay allowed for highly accurate identification of the eight most-common beta-thalassemia mutations in homozygous normal control, carrier, and eight heterozygous carrier mixtures, as well as the diagnosis of a high-risk family. The results demonstrated a flexible strategy for rapid identification of clustering SNPs in HBB with a high degree of accuracy and specificity. It can be adapted easily for high-throughput diagnosis of various hereditary diseases or to establish family heritage databases for clinical applications.

Base Sequence↗

PubMed2006

Mononeuritis multiplex as a presenting feature of Wegener granulomatosis: a case report.

We report the case of a man presenting with clinical features of a mononeuritis multiplex, a perinuclear-ANCA (p-ANCA), and a renal biopsy suggestive of Wegener granulomatosis (WG). We wish to highlight this case as a learning point for clinicians as WG rarely presents in this form, and can be easily overlooked as a cause of mononeuritis multiplex.

Gait Disorders, Neurologic↗

PubMed2006

Multiplex PCR-ELISA for direct detection of MRSA in nasal swabs advantageous for rapid identification of non-MRSA carriers.

In the study presented here 251 nasal swabs obtained from medical staff were directly investigated for MRSA using a commercial multiplex PCR system in parallel with conventional culture methods to determine the usefulness of PCR for rapid screening. Both methods identified 3.2% (8/251) of specimens as MRSA-positive; one sample was culture-positive only, and three were PCR-positive only. PCR correctly identified 215 of 239 (90%) negative swab samples, but one sample with weak cultural growth was not detected and was therefore considered false negative. The comparative sensitivity of culture versus PCR was 75% (9/12) versus 91.6% (11/12). Although PCR had a low positive predictive value (31.4%) its negative predictive value was high (99.5%). The results of this study indicate the multiplex PCR is suitable for the rapid identification of MRSA-negative individuals directly from nasal swabs in populations with a low MRSA prevalence, but positive results need to be confirmed by culture.

Enzyme-Linked Immunosorbent Assay↗

PubMed2000

Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse-transcriptase polymerase chain reaction.

The aim of this study was to generate urgently needed data on respiratory pathogens in German children using an economical and efficient tool. Nasopharyngeal aspirates of hospitalized children 0-16 years of age with an acute respiratory tract infection were tested by a nine-valent multiplex reverse-transcriptase polymerase chain reaction. Of 1281 children, 449 (35%) had an acute respiratory tract infection caused by at least one of the organisms studied; there were 29 cases of dual infection. At least 34-42% of severe acute respiratory tract infections in children under 5 years of age were caused by viruses. In children over 5 years of age, this proportion was 23% (P<0.001). Infection during the first 2 years of life was most frequently due to respiratory syncytial virus (n = 162 cases). Parainfluenza virus type 3 (n = 22) and type 1 (n = 14) were detected almost exclusively in children under 5 years of age. Influenza A (n = 90) and adenoviruses (n = 98) were prevalent in all age groups. The frequency of influenza B virus isolation (n = 17) rose significantly after the age of 5 years. Mycoplasma pneumoniae infection (n = 24 cases, 5.2%) was most frequent in 5- to 16-year-old patients. Only one case of Chlamydia pneumoniae infection was found. Since the distribution of pathogens within the different types of lower respiratory tract infections is very similar, it seems that host factors determine which form of lower respiratory tract infection develops in an individual patient. The multiplex reverse-transcriptase polymerase chain reaction may, in the future, become an important tool for epidemiological studies as well as for individual diagnosis.

Adolescent↗

PubMed2001

Usefulness of adding multiplex nested-polymerase chain reaction assay of cerebrospinal fluid samples to routine diagnostic testing for herpesvirus encephalitis.

The present study was conducted to assess the usefulness of adding the multiplex nested-polymerase chain reaction assay of cerebrospinal fluid samples to routine diagnostic testing for herpesvirus encephalitis and to monitor the efficacy of therapy. Cerebrospinal fluid samples from 45 patients with presumed herpesvirus encephalitis were tested for herpes simplex virus, varicella-zoster virus, cytomegalovirus, human herpesvirus 6, and Epstein-Barr virus. Ten of the 45 patients were positive for a virus using the polymerase chain reaction assay: herpes simplex virus (n=5), Epstein-Barr virus (n=3), and herpes simplex virus plus the Epstein-Barr virus (n=2). Cerebrospinal fluid from two patients who had undergone acyclovir therapy gave negative results. Analysis of cerebrospinal fluid by multiplex polymerase chain reaction can be useful for establishing an accurate diagnosis and as a marker of the efficacy of therapy.

Adult↗

PubMed2001

Rapid and simultaneous identification of body parts from the morphologically similar sharks Carcharhinus obscurus and Carcharhinus plumbeus (Carcharhinidae) using multiplex PCR.

Many commercially exploited carcharhinid sharks are difficult to identify to species owing to extensive morphological similarities. This problem is severely exacerbated when it comes to identifying detached shark fins, and the finless and headless shark carasses typically sold in markets. To assist in the acquisition of urgently needed conservation and management data on shark catch and trade, we have developed a highly streamlined approach based on multiplex polymerase chain reaction (PCR) that uses species-specific primers derived from nuclear ribosomal ITS2 sequences to achieve rapid species identification of shark body parts. Here we demonstrate the utility of this approach for identifying fins and flesh from two globally distributed, morphologically very similar carcharhinid sharks (Carcharhinus obscurus and Carcharhinus plumbeus) intensively targeted in fisheries worldwide, and often confused for each other even as whole animals. The assay is conducted in a 4-primer multiplex format that is structured to simultaneously achieve the following efficiency and cost-reduction objectives: it requires only a single-tube amplification reaction for species diagnosis, it incorporates an internal positive control to allow detection of false-negative results, and it is novel in that it allows species identification even when DNAs from two species are combined in the same tube during the PCR reaction. The latter innovation reduces the required effort for screening a set of unknown samples by 50%. The streamlined approach illustrated here should be amenable for use in a shark conservation and management context where large numbers of samples typically need to be screened; the approach shown may also provide a model for a rapid diagnostic method applicable to species identification in general.

Journal Article↗

PubMedDate not supplied

Direct multiplex assay of enzymes in dried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders.

Tandem mass spectrometry is currently used in newborn screening programmes to quantify the level of amino acids and acylcarnitines in dried blood spots for detection of metabolites associated with treatable diseases. We have developed assays for lysosomal enzymes in rehydrated dried blood spots in which a set of substrates is added and the set of corresponding enzymatic products are quantified using tandem mass spectrometry with the aid of mass-differentiated internal standards. We have developed a multiplex assay of the set of enzymes that, when deficient, cause the lysosomal storage disorders Fabry, Gaucher, Hurler, Krabbe, Niemann-Pick A/B and Pompe diseases. These diseases were selected because treatments are now available or expected to emerge shortly. The discovery that acarbose is a selective inhibitor of maltase glucoamylase allows the Pompe disease enzyme, acid alpha-glucosidase, to be selectively assayed in white blood cells and dried blood spots. When tested with dried blood spots from 40 unaffected individuals and 10-12 individuals with the lysosomal storage disorder, the tandem mass spectrometry assay led to the correct identification of the affected individuals with 100% sensitivity. Many of the reagents needed for the new assays are commercially available, and those that are not are being prepared under Good Manufacturing Procedures for approval by the FDA. Our newborn screening assay for Krabbe disease is currently being put in place at the Wadsworth Center in New York State for the analysis of approximately 1000 dried blood spots per day. Summary We have developed tandem mass spectrometry for the direct assay of lysosomal enzymes in rehydrated dried blood spots that can be implemented for newborn screening of lysosomal storage disorders. Several enzymes can be analysed by a single method (multiplex analysis) and in a high-throughput manner appropriate for newborn screening laboratories.

Blood Specimen Collection↗