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PubMed2002

Frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms in a Brazilian population.

The glutathione S-transferase (GST) family of enzymes has a vital role in phase II of biotransformation of environmental carcinogens, pollutants, drugs and other xenobiotics. GSTs are polymorphic, with the type and frequency of polymorphism being ethnic dependent. Polymorphisms in GST genes have been shown to be associated with susceptibility to disease and disease outcome. We determined the frequencies of GSTM1, GSTT1 and GSTP1 polymorphisms in 591 volunteers who had been residents of Rio de Janeiro for at least six months. Blood was collected and DNA extracted by proteinase K/SDS digestion. Information about social habits and health problems was also recorded. GSTM1 and GSTT1 polymorphisms were analyzed by a PCR-Multiplex procedure, whereas GSTP1 polymorphism was analyzed by PCR-RFLP. We found that 42.1% (48.9% of whites and 34.2% of non-whites) of the individuals had the GSTM1 null genotype, whereas 25.4% (25.1% of whites and 25.7% of non-whites) had the GSTT1 null genotype. The genotypic distribution of GSTP1 was 49.7% I/I, 38.1% I/V, and 12.2% V/V, whereas the allelic frequencies were 0.69 for the Ile allele, and 0.31 for the Val allele. The frequencies of GST polymorphisms in this Brazilian population were found to be different from those observed in other populations, particularly of other South American countries.

Adolescent↗
PubMed2004

Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR.

A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome.

Animals↗
PubMed1998

Accurate, high-throughput "snapshot" detection of hMLH1 mutations by two-dimensional DNA electrophoresis.

The human genome and related projects have resulted in the isolation of a rapidly growing number of genes that cause susceptibility to human cancer. With rare exception, these genes are large and have disease-associated mutations scattered along the length of the genes. Therefore, the development of accurate and cost-efficient mutation detection tests that can scan entire genes singly or in combination is warranted. hMLH1, encoding a mismatch repair enzyme, is a susceptibility gene for hereditary nonpolyposis colon cancer syndrome. This gene comprises 19 exons; mutations are scattered, typical of many susceptibility genes. Here, we present a strategy that combines extensive PCR multiplexing and two-dimensional DNA electrophoresis (Two-Dimensional Gene Scanning, TDGS) to scan accurately for mutations that lie within the exons and splice junctions of hMLH1. All target fragments, designed to have optimal melting characteristics, were prepared in a two-stage PCR--a four-plex long-distance PCR followed by short PCR in two multiplex groups of 10 and 11 amplicons. The mixture of amplicons was subjected to two-dimensional electrophoresis: separation by size in the first dimension and by melting characteristics in the second. Using this design, 41 samples containing known hMLH1 sequence variants or no alterations were blindly subjected to TDGS. All mutations were detected; there were no genuine false-positive or false-negative results. These results confirm that TDGS is a generally applicable, rapid, accurate, and reproducible mutation detection technology that would serve large-scale molecular epidemiologic studies as well as clinical molecular diagnostic purposes.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Reliability of preimplantation diagnosis for single gene disorders.

Reliability of preimplantation genetic diagnosis (PGD) depends on controlling one of the most important limitations of single cell PCR, undetected allele drop out (ADO), which may lead to misdiagnosis. To avoid this we introduced mutation analysis simultaneously with linked polymorphic markers, pre-selecting only those embryos whose unaffected status could be confirmed by at least one linked polymorphic marker. We applied this strategy for testing 1047 oocytes, from which 237 unaffected ones were pre-selected for transfer back to patients, resulting in 34 unaffected pregnancies and birth of 23 healthy children. Embryos originating from mutant oocytes and those with insufficient marker information were followed up by multiplex PCR to confirm single cell PCR diagnosis. Of 75 (8.5%) detected ADO, only seven (under 1%) were missed in the actual PGD, demonstrating high reliability of PGD (98%) based on multiplex single cell PCR.

Alleles↗
PubMed2002

Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis.

A fast and reliable Multiplex-PCR assay was established to identify the species Lactobacillus johnsonii. Two opposing rRNA gene-targeted primers have been designed for this specific PCR detection. Specificity was verified with DNA samples isolated from different lactic acid bacteria. Out of 47 Lactobacillus strains isolated from different environments, 16 were identified as L. johnsonii by PCR. The same set of strains was investigated with five alternative molecular typing methods: enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR), amplified fragment length polymorphism, single triplicate arbitrarily primed PCR, and pulsed-field gel electrophoresis in order to compare the discriminatory power of these methods. The reported data strongly support the highly significant heterogeneity among all L. johnsonii isolates, potentially linked to their origin of isolation. The use of species-specific primers as well as rapid and highly powerful PCR-based molecular typing tools (namely ERIC- and REP-PCR techniques) should be respectively envisaged for identifying, differentiating and monitoring L. johnsonii strains from various environmental samples, for product monitoring, for species tracing in clinical studies as well as bacterial profiling of various microecological or gastrointestinal environments.

Bacterial Typing Techniques↗
PubMed2005

Abnormalities of the P53, MDM2, BCL2 and BAX genes in acute leukemias.

Abnormalities of the P53 network have been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The purpose of this study was to define P53 gene mutations, to detect MDM2 gene amplification and to estimate mRNA expression of P53, MDM2, BCL2 and BAX genes in patients with ALL and AML. Twenty-five patients with ALL and 65 patients with AML, both recently diagnosed, were included into this study. Exons 5-8 of the P53 gene with flanking intronic sequence were amplified by the polymerase chain reaction (PCR) method and subjected to mutation screening by single-strand conformation polymorphism analysis (SSCP). Mutation of the P53 gene was found in one patient of the 25 with ALL and in five patients of the 65 with AML. Sequence analysis was subsequently performed. One mutation in intronic sequence in ALL and four missense mutations and one silent nucleotide substitution in AML were identified. Amplification of MDM2 gene was detected by multiplex-PCR analysis in only one sample from patient with ALL, but was not observed in any case of AML. To gain further insight into the role of P53 network in the evolution of acute leukemias, the P53, MDM2, BCL2 and BAX mRNAexpressions in portion samples from patients with ALL and AML were analyzed using multiplex RT-PCR. Although a low frequency of molecular disturbances of the P53 and the MDM2 genes was detected in this study, there was a high percentage of cases with increased mRNA level of P53 and MDM2. A high frequency of BCL2 mRNA overexpression and a relatively low frequency of BAX mRNA overexpression detected in both analyzed leukemias in this study, indicate that altered transcription of these genes may be involved in leukemogenesis.

Adolescent↗
PubMed1995

A method for the purification and recovery of genomic DNA from an HLA DQA1 amplification product and its subsequent amplification and typing with the AmpliType PM PCR Amplification and Typing Kit.

DNA from plucked single hairs from ten individuals was extracted by two different methods and subsequently amplified and typed using the AmpliType HLA DQ alpha Forensic DNA Amplification and Typing Kit. The remaining untyped portions of the DQA1 amplification products were stored refrigerated or frozen for two weeks and subsequently purified using Centricon 100 microconcentrators. Genomic DNA was recovered from the DQA1 amplification PCR and used again as a template for a subsequent multiplex PCR. Twenty microL of each retentate were amplified and typed with the AmpliType PM PCR Amplification and Typing Kit. All typing results were consistent with DQA1 and PM results of control hairs and reference blood samples from the donors and all results were consistent with those obtained when the samples were typed solely for PM. The DQA1-Centricon 100-PM approach is useful when the genomic DNA from an evidentiary sample has been used completely for HLA-DQA1 typing, so that only the amplified product is remaining. The typing of five more genetic markers can be achieved from a HLA-DQA1 sample, so additional information for identification purposes could be provided. However, genomic DNA as well as the DQA1 product are recovered and the latter will also serve as a template in the subsequent PM amplifications. Therefore there will be more DQA1 product after the PM amplification than would be expected when only genomic DNA was used as a template. Thus certain practices should be considered when reading the types from PM probe strips if this DQA1-Centricon 100-PM approach is used.

DNA↗
PubMed2001

Simultaneous detection of influenza viruses A and B using real-time quantitative PCR.

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

Follow-Up Studies↗
PubMed1998

Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells.

In many cases of human cancer, the appearance of hypersialylated glycan structures is related to a precise stage of the disease; this may depend on altered regulation of one or more sialyltransferases genes. Since several distinct sialyltransferase enzymes arising from different unique genes transfer sialic acid residues in the same linkage onto the same acceptor, it is impossible to precisely determine which enzyme is involved in the observed phenotype based on enzymatic assays. We have developed a very sensitive and highly reproducible multiplex reverse transcriptase-polymerase chain reaction technique in order to monitor the expression of four human sialyltransferases genes ST6Gal I, ST3Gal I, ST3Gal III and ST3Gal IV in small cell samples. Multiplex PCR amplification using specific primers for each sialyltransferase and detection of amplification products by polyacrylamide gel electrophoresis is a method that is fast and easy to handle and has proven to be useful for establishing sialyltransferase patterns of expression in breast immortalized cell line HBL100 as well as in breast cancer cell lines MCF-7/6, MCF-7/AZ and MDA.

Breast Neoplasms↗
PubMed2005

Rapid identification of human adenovirus types 3 and 7 from respiratory specimens via multiplex type-specific PCR.

The rapid diagnosis of human adenovirus (Ad) infection is crucial for the timely recognition of epidemics. Moreover, identification of the serotypes known to cause serious disease can be helpful in therapeutic intervention. A multiplex PCR assay was developed for the rapid detection of adenovirus type 3 (Ad3) and Ad7 directly from clinical specimens. For this assay, three primer pairs (primers were based on the conserved and hypervariable regions of the hexon) were designed in order to simultaneously amplify all adenoviral serotypes and discriminate between Ad3 and Ad7. In our preliminary analysis, this multiplex PCR assay generated amplicons of the consensus primers from all 106 adenoviral isolates of diverse serotypes and proved able to correctly identify Ad3 and Ad7. This assay was subsequently applied to the detection of Ad3 and Ad7 in respiratory specimens. Among the 127 nasal aspirates from which an adenovirus was grown, the sensitivity with which any serotype could be detected was 91% (115/127). Two of the 53 nasal aspirates which did not grow Ads yielded adenovirus-specific bands, which were confirmed by sequencing analysis. Among the 115 specimens which produced common adenoviral bands, the sensitivity with which Ad3 could be detected was 93% (26/28), and the sensitivity with which Ad7 could be detected was 100% (35/35). Five out of the 115 specimens were proved to harbor more than one type of Ad via sequencing analysis of the amplicons, suggesting mixed infection with at least two different serotypes. In conclusion, this multiplex PCR system can be utilized in the rapid identification of Ad3 and Ad7 directly from clinical specimens. Furthermore, this method constitutes a diagnostic strategy for the detection of coinfection by different Ad serotypes.

Adenovirus Infections, Human↗
PubMed2006

Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain.

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclaved wheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and <54 CFU/mL, respectively, in the initial sample. For L. monocytogenes, an improved detection limit of <62 CFU/mL was achieved using singleplex PCR. For autoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichment step. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.

Animals↗
PubMed2004

[A combined assay of multiplex RT-PCR and karyotypic analysis in childhood acute lymphoblastic leukemia].

OBJECTIVE: To study the value of combination assay of multiplex RT-PCR and karyotypic analysis in the diagnosis and classification of childhood acute lymphoblastic leukemia (ALL). METHODS: Fifty cases of childhood ALL patients were studied by multiplex RT-PCR in combination with R or G banding karyotype analysis. RESULTS: Of the 50 childhood ALL patients, 18 (36.0%) carried 11 types of fusion genes including E2A/PBX1, TEL/AML1, TLS/ERG, MLL/AF4, MLL/AF9, MLL/AF10, MLL/AFX, MLL/AF6, MLL/ELL, TAL1D, and HOX11, revealed by multiplex RT-PCR, and in 48 cases, 24 (57.1%) had chromosome abnormalities. Among the latter, numeral chromosome abnormalities and chromosome deletions accounted for 75.0% (18/24), while translocations 25.0% (6/24). The multiplex RT-PCR in combination with chromosome analysis could detect genetic abnormalities in 70% (35/50) of childhood ALL. CONCLUSIONS: Multiplex RT-PCR combined with chromosome analysis can enhance the detection rate of genetic abnormalities in childhood ALL. It provides reliable evidence for the diagnosis, classification and prognosis.

Adolescent↗
PubMed2000

Detection of PCR products using PNA strand invasion.

The unique ability of homopyrimidine peptide nucleic acid (PNA) to strand invade homopurine sites of duplex DNA offers a potential alternative to existing techniques for rapid detection of PCR products. From gel shift studies, PNA was found to specifically strand invade homopurine sites that had been incorporated into an amplicon during the PCR cycle. This was achieved by adding a homopyrimidine sequence to the 5'-terminus of a PCR primer. The position of the strand invasion sites at the termini of the DNA duplex offers kinetic advantages for PNA strand invasion, since the termini of DNA duplexes are known to be unwound. This unwound state was demonstrated using a novel assay that determined single-stranded regions within the amplicon. The presence of the PNA moiety in strand invasion complexes was confirmed by a novel electroblot, an Enzyme Linked Nucleic Acid assay and by an increase in stability as demonstrated by T(m)studies with the Idaho RapidCycler. Since the strand invasion sites can be controlled through selection of the homopurine sequence there is great flexibility for designing strand invasion motifs unique to a particular PCR amplicon, thus providing a huge potential for differentiating and detecting multiplex PCR products.

Base Sequence↗
PubMed2006

Molecular diagnosis of alpha-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis.

The aim of the study was to set up an automatic molecular diagnostic method for deletional alpha-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by dissociation curve (DC) analysis were used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles (alpha alpha or alpha(T)alpha), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the dissociation curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon > or = cutoff values. Molecular diagnosis of alpha-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles were 82.5 +/- 1 degrees Celsius, 82.8 +/- 1 degrees Celsius, 81.5 +/- 1 degrees Celsius, and 83.0 +/- 1 degrees Celsius, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The C(T) VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10(5) approximately 10(0). Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 alpha-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of alpha-thalassemia.

Alleles↗
PubMed2001

Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR.

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding approximately 500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.

Animals↗
PubMed1996

Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis.

We have developed a semiautomated approach to amplify 25 exons of the dystrophin gene using two fluorescent multiplex PCR assays which detect over 98% of reported deletions and 90% of duplications causing Duchenne/Becker muscular dystrophy. The 5' multiplex detects 11 exons from the proximal deletion hotspot of the gene while the 3' multiplex detects 14 exons from the central deletion hotspot. The PCR products are accurately sized and quantified by a fluorescent DNA sequencer after only 18 cycles of amplification. The amount of product amplified from each exon in a multiplex is divided by that from each of the other exons, and this ratio is compared with those from control samples to obtain a series of dosage quotients (DQ), from which the copy number of each exon is determined. No overlap was observed between the DQ values obtained from single and double copy loci. The assays can be used to screen both affected males and at risk female relatives for a mutation. The method has been evaluated as a female carrier test by conducting a blind trial on 150 coded samples. Sixty-three deletion carriers, two duplication carriers, and 84 normal female controls were all correctly identified, showing that carrier diagnosis is possible even in families where the nature of the mutation is unknown. Additionally the analysis showed a non-pathogenic duplication involving the muscle specific promoter and exon 1. Together these two multiplex assays detect over 70% of all mutations in the dystrophin gene, greatly simplifying and partly automating molecular diagnosis in Duchenne and Becker muscular dystrophy.

Automation↗
PubMed2004

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses.

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.

Child, Preschool↗