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Results for “Multiplex PCR”

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PubMed2006

[Detecting the haplotype of three Y-STR loci by fluorescent multiplex amplification].

OBJECTIVE: To establish a fluorescent multiplex PCR system for typing Y-STR loci Y-GATA-A7.1, DYS456 and DYS443, and investigate their haplotype frequencies in Chinese Han population. METHODS: 203 unrelated males of Han population living in Zhengzhou were typed by fluorescent multiplex amplification system and ABI 3100 genetic analyzer. RESULTS: In Zhengzhou Han population, 5,6 and 6 different alleles were observed for Y-GATA-A7.1, DYS456 and DYS443 loci, and their gene diversity (GD) were 0.669 2, 0.583 9 and 0.705 3 respectively. A total of 44 different haplotypes formed by these three loci was identified and the haplotype diversity (HD) reached 0.952 3. CONCLUSION: The fluorescent multiplex system for these three Y-STR loci will be very powerful for forensic individual identification and paternity testing in Chinese Han population.

Alleles↗

PubMed2004

New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

Anti-Bacterial Agents↗

PubMed1991

Workshop overview: new molecular techniques in genome analysis.

An overview of the application of various molecular techniques to the analysis of genomic DNA is presented. For the analysis of small-scale changes, the polymerase chain reaction (PCR), colony probe hybridization, mismatch hybridization, and denaturing gradient gel electrophoresis (DGGE) are providing information on mutations within prokaryotic and eukaryotic genes. For large-scale changes, fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), Southern blotting, multiplex PCR, hybridization of linked probes, and restriction enzyme mapping are permitting analysis of genomic alterations that are larger than point mutations but below the resolution of standard cytogenetic analysis. Many of these techniques, either alone or in combination, produce DNA that can be subjected to DNA sequence analysis, which provides the most detailed information regarding genomic changes.

Chromosome Aberrations↗

PubMed2005

Detection of non-pylori Helicobacter species in "Helicobacter heilmannii"-infected humans.

BACKGROUND: A small proportion of patients suffering from chronic active gastritis are diagnosed with gastric Helicobacter species other than Helicobacter pylori. Circumstantial evidence has suggested that these bacteria, also referred to as "Helicobacter heilmannii"-like organisms (HHLO), may be transmitted through animals. The isolation of a Helicobacter bizzozeronii strain from a human patient confirmed this hypothesis. It was the aim of the present study to assess the presence of animal Helicobacter species and H. pylori in humans infected with HHLO, as diagnosed by histology. METHODS: Paraffin-embedded gastric biopsy specimens of 108 HHLO-infected patients (42 women and 66 men) from three clinical centers were screened for the presence of animal gastric Helicobacter species by polymerase chain reaction (PCR), using assays targeting the 16S rDNA region of the three known canine and feline helicobacters (H. bizzozeronii, H. salomonis and H. felis), "Candidatus H. suis", and "Candidatus H. bovis". In addition, the presence of H. pylori was evaluated by multiplex PCR analysis. RESULTS: In 63.4% of the stomachs (64/101) classification of the Helicobacter infection into the above mentioned groups was achieved. Non-pylori Helicobacter species commonly colonizing the stomachs of cats and dogs were found in 48.5% (49/101) of the patients. Fourteen (13.9%) samples tested positive for "Candidatus H. suis", and "Candidatus H. bovis" was demonstrated in 1 (0.9%) patient. The presence of H. pylori was established in 13 patients (12.9%). Eleven stomachs (10.9%) were infected with at least two different Helicobacter species. CONCLUSIONS: This study identifies animal Helicobacter species in the stomach of a large series of HHLO-infected patients, which may have clinical implications in a subset of patients with gastric disease.

Adolescent↗

PubMed2004

Heterogeneous t(4;11) fusion transcripts in two infants with acute lymphoblastic leukemia.

An RT-PCR assay detected the t(4;11) translocation in two infants with acute lymphoblastic leukemia (ALL). Case P76 was a 10-month-old, female infant, who presented with a WBC of 137.4 x 10(9)/l and a pre-pre-B ALL immunophenotype. Case P120 was a 6-month-old female infant, with a WBC > 615 x 10(9)/l and a pre-pre-B ALL immunophenotype. RT-PCR of cDNA from both these cases generated a 656 bp and a 542 bp respectively, which sequencing confirmed as t(4;11) fusion transcripts. The primers and conditions selected for this assay are compatible with a one-step multiplex PCR for the main translocations in childhood ALL.

Base Sequence↗

PubMed2004

A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus types A and B.

A multiplex reverse-transcription polymerase chain assay (mRT-PCR) was developed, based on primers designed to distinguish the A and B types of beet necrotic yellow vein virus (BNYVV). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established detection method based on single strand conformation polymorphisms (SSCPs). An area of the 'triple gene block' region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. The A type assay produced a single 324 base-pair RT-PCR fragment when positive samples were amplified. The B type assay produced a 178 base-pair product from positive samples. No amplification was observed from healthy Chenopodium quinoa or sugar beet plants and from plants infected by others sugar beet soil-borne viruses. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. The results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples.

Base Sequence↗

PubMed1997

Detection of Francisella tularensis in ulcers of patients with tularemia by PCR.

The diagnosis of human cases of tularemia is usually confirmed by the demonstration of an antibody response to Francisella tularensis, which occurs about 2 weeks after the onset of disease. Due to a high risk of infection in the laboratory, cultivation of the causative agent tends to be avoided. During an outbreak in Sweden, the use of PCR for diagnosing the ulceroglandular form of tularemia was evaluated. Extraction and preparation of F. tularensis DNA from swab samples from the wounds of patients with tularemia involved the use of the nuclease inhibitor guanidine thiocyanate. The DNA was detected by multiplex PCR targeting the 16S rRNA gene and a 17-kDa lipoprotein gene of F. tularensis. In 29 of 40 (73%) patients with serologically confirmed tularemia, F. tularensis DNA was successfully amplified. Considering the limitations of current diagnostic procedures, PCR may become useful for the early diagnosis of tularemia.

Adolescent↗

PubMed2003

Hexaplex PCR for rapid detection of virulence factors.

PCR technology has emerged as a basic tool in biological research and in the detection of infectious organisms. It has the potential to provide information on a number of toxins and virulence factors, as well as allowing species identification of pathogens. Multiplex PCR assays are becoming prevalent for the simultaneous detection of toxins, virulence factors and pathogens in clinical and environmental specimens. This review will discuss the hexaplex PCR assay, its application and future directions.

Animals↗

PubMed2003

Easy, accurate and reliable screening for SNPs by ion pair/reverse phase HPLC: simultaneous detection of factor V G1691A, prothrombin G20210A and methylenetetrahydrofolate reductase C677T variants.

We have set up allele specific PCR in combination with high-performance liquid chromatography as a method to simultaneously detect three SNPs, factor V (G1691A), factor II/prothrombin (G20210A) and methylenetetrahydrofolate reductase (C677T) genes, all suspected to be inherited risk factors for deep vein thrombosis. Allele specific multiplex PCR technique was used to generate PCR products of different sizes which were specific for each genotype analyzed in this study. PCR products were analyzed on the WAVE Nucleic Acid Analysis platform by means of a gradient ion pair/reverse phase chromatography without any further purification. This method allows concurrent genotyping of the PCR products of the three mutations within 8 minutes, clearly reduces working time and costs in a routine laboratory, and can easily be adapted to the analysis of any other SNP or the combination of different other SNPs. The assay established here has been validated on more than a hundred samples and gave correct and highly reproducible intra- and interday results, allowing the application of this easy, accurate and reliable test in routine settings and in clinical trials and epidemiological surveys.

Chromatography, High Pressure Liquid↗

PubMed2004

Detection of Cyclospora cayetanensis in animal fecal isolates from Nepal using an FTA filter-base polymerase chain reaction method.

Cyclospora cayetanensis is an emerging protozoan parasite capable of causing a protracted diarrheal illness in both immunocompromised and immunocompetent individuals. Ingestion of fresh produce and water sources contaminated with mature sporulated oocysts results in acquisition of cyclosporiasis. Currently, no animal model exists for the study of this pathogenic parasite and the only confirmed reservoir host for C. cayetanensis in nature is humans. Previously, Cyclospora-like oocysts had been detected by microscopy in several animals including non-human primates. However, their phylogenetic relationship to C. cayetanensis remained uncertain due to the limited availability of molecular techniques to differentiate and speciate these isolates. In the present study, we examined a series of fecal isolates obtained from dogs, chickens, and monkeys collected between May and September 2002 from several geographic regions of Nepal. All samples were examined by microscopy and a polymerase chain reaction (PCR) for the presence of C. cayetanensis. Both microscopic and conventional PCR/restriction fragment length polymorphism (RFLP) analysis demonstrated the presence of Cyclospora sp. in the fecal samples of two dogs, one chicken, and one monkey. Application of a species-specific multiplex PCR assay confirmed the presence of both Eimeria sp. and C. cayetanensis in the positive chicken sample and only C. cayetanensis in the dog and monkey samples. However, in the absence of tissue analysis, the assignment of these animals as a natural reservoir host for C. cayetanensis remains to be determined.

Animals↗

PubMed2005

Molecular genetic confirmatory testing from newborn screening samples for the common African-American, Asian Indian, Southeast Asian, and Chinese beta-thalassemia mutations.

beta-Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with beta-thalassemia. Our purpose was to develop single-tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for beta-thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic-specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for beta-thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African-American descent. Specimens from African-American neonates were tested for two mutations (-88 and -29); Asian Indians for five mutations (IVSI-1, IVSI-5, codons (Cd) 41/42, Cd 8/9, and 619-bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, -28, IVSII-654, Cd 71/72, IVSI-5, and IVSI-1). We identified each of these beta-thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with beta-thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic-specific beta-thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation.

Asian People↗

PubMed2004

Multiplexed RT-PCR for streamlined detection and separation of barley and cereal yellow dwarf viruses.

Two novel multiplexed RT-PCR assays that can efficiently detect and distinguish among different barley and cereal yellow dwarf viruses (B/CYDVs) are described. The basic multiplex can produce two fragments simultaneously, a approximately 830-bp fragment indicating the presence of the BYDV-PAV, BYDV-MAV, or BYDV-SGV viruses and a approximately 372-bp fragment indicating the presence of the CYDV-RPV, BYDV-RMV, or BYDV-GPV viruses. The enhanced multiplex produces two additional fragments, which further differentiate between BYDV-PAV, BYDV-MAV, and BYDV-SGV. These assays fulfill the critical need for a streamlined diagnostic procedure for B/CYDVs that can be cost-effectively applied to large numbers of small samples. The assays are useful not only in the basic diagnosis of B/CYDVs, but also for studies examining the ecological roles of B/CYDVs in natural systems and for longer-term epidemiological studies of grasses and cereals.

DNA, Complementary↗

PubMed1998

Detection of methicillin resistance in coagulase-negative staphylococci initially reported as methicillin susceptible using automated methods.

Reliable detection of methicillin resistance in coagulase-negative staphylococci (CNS) is required for appropriate therapy of serious infections from these pathogens. To determine the most accurate method of measuring methicillin resistance in CNS initially reported as methicillin susceptible by automated methods, we compared mecA detection by polymerase chain reaction (PCR) with phenotypic methods. One hundred eighty-eight blood culture isolates of CNS that were initially reported as susceptible to methicillin using commercial methods (Vitek or MicroScan) were tested by agar dilution, disk diffusion, oxacillin salt agar screen plate, and a multiplex PCR assay using primer sets for mecA and 16S rRNA. Sixteen isolates (8.5%) previously reported as methicillin susceptible by automated methods contained the mecA gene. MICs of these isolates ranged from 0.5 microgram/mL to > or = 128 micrograms/mL. Ten of these isolates had MICs equal to or below the NCCLS breakpoint of 2 micrograms/mL. Six of the 10 isolates (4 with MICs of 0.5 microgram/mL and 2 with MICs of 2 micrograms/mL) did not grow on any of the oxacillin screen plates after 48 h of incubation at 30 degrees C or 35 degrees C. All six isolates were induced to grow in the presence of oxacillin at 128 micrograms/mL by serial passaging on plates containing increasing concentrations of antibiotic. Retesting with MicroScan and Vitek detected methicillin resistance in 7 and 10 isolates, respectively. Disk diffusion testing with incubation for 48 h proved to be the next best method after PCR for detection of methicillin resistance (15 of 16 isolates). Commercial automated methods and some methods recommended by National Committee for Clinical Laboratory Standards may not detect methicillin resistance in CNS that carry the mecA gene and have MICs just below breakpoint.

Bacterial Proteins↗

PubMed2006

Polymorphisms in the glucocerebrosidase gene and pseudogene urge caution in clinical analysis of Gaucher disease allele c.1448T>C (L444P).

BACKGROUND: Gaucher disease is a potentially severe lysosomal storage disorder caused by mutations in the human glucocerebrosidase gene (GBA). We have developed a multiplexed genetic assay for eight diseases prevalent in the Ashkenazi population: Tay-Sachs, Gaucher type I, Niemann-Pick types A and B, mucolipidosis type IV, familial dysautonomia, Canavan, Bloom syndrome, and Fanconi anemia type C. This assay includes an allelic determination for GBA allele c.1448T>C (L444P). The goal of this study was to clinically evaluate this assay. METHODS: Biotinylated, multiplex PCR products were directly hybridized to capture probes immobilized on fluorescently addressed microspheres. After incubation with streptavidin-conjugated fluorophore, the reactions were analyzed by Luminex IS100. Clinical evaluations were conducted using de-identified patient DNA samples. RESULTS: We evaluated a multiplexed suspension array assay that includes wild-type and mutant genetic determinations for Gaucher disease allele c.1448T>C. Two percent of samples reported to be wild-type by conventional methods were observed to be c.1448T>C heterozygous using our assay. Sequence analysis suggested that this phenomenon was due to co-amplification of the functional gene and a paralogous pseudogene (PsiGBA) due to a polymorphism in the primer-binding site of the latter. Primers for the amplification of this allele were then repositioned to span an upstream deletion in the pseudogene, yielding a much longer amplicon. Although it is widely reported that long amplicons negatively impact amplification or detection efficiency in recently adopted multiplex techniques, this assay design functioned properly and resolved the occurrence of false heterozygosity. CONCLUSION: Although previously available sequence information suggested GBA gene/pseudogene discrimination capabilities with a short amplified product, we identified common single-nucleotide polymorphisms in the pseudogene that required amplification of a larger region for effective discrimination.

Alleles↗

PubMed2000

Plasmodium falciparum: detection and strain identification of Indian isolates by polymerase chain reaction.

The polymerase chain reaction (PCR) was employed for detection and strain identification of P. falciparum in a comparative field study of Indian isolates. The primers were selected from highly conserved regions flanking the variable, tandemly repeated regions of highly polymorphic cell surface antigens, major merozoite surface antigen-1 (MSP-1), major surface antigen-2 (MSP-2), circumsporozoite surface antigen (CSP) and ring-infected erythrocyte surface antigen (RESA). Out of the 52 microscopically positive P. falciparum infected field samples, 47 samples were positive by PCR. Variation in the size of the amplified products was observed using MSP-1, MSP-2 specific primers respectively in different field isolates of P. falciparum, but CSP and RESA did not exhibit any variation in size of the amplified product. The multiplex PCR results demonstrated that amplified products from these surface antigens vary in size and there is a specific pattern for each strain and this could be utilized to identify a particular field isolate. One P. falciparum infected field sample detected by the above PCR method was found to be a mixed infection by two different strains. Five microscopically positive P. vivax infeced samples were also analyzed by PCR method using P. falciparum cell surface antigen (MSP-2) specific primers. PCR results showed one P. vivax infected sample was positive when P. falciparum specific primers were used, this could be due to inaccurate and reduced limit of detection of Plasmodial species by microscopic examination.

Animals↗

PubMed2001

Predominant human rotavirus genotype G1P[8] infection in infants and children in Bangkok, Thailand.

Human rotavirus is the major etiologic agent of infantile diarrhea on a worldwide scale. In this study, rotaviruses were detected by reverse-transcription PCR in 42 of 83 stool specimens from children below the age of 3 years with acute diarrhea in Bangkok, Thailand, between November 1998 and August 1999. G and P types of all samples were characterized by restriction endonuclease analysis (REA) and multiplex PCR typing assay, respectively. Strain G1P[8] (76.1%) was the predominant type, followed by G1P[6] (2.4%). Strain G1 combined with mixed P[8]/P[6] was identified in 2 specimens (4.8%) and 7 untypeable G strains (16.7%) were observed. This information on the circulating G and P combinations should be useful for understanding the epidemiology of human rotavirus in Bangkok, Thailand.

Acute Disease↗

PubMed2002

[Sharper diagnostic tool the future promise of laser-assisted microdissection].

The paper describes the use of laser-assisted microdissection to retrieve microscopically defined cell populations including single cells from tissue sections for subsequent analysis of genomic DNA and mRNA. A general background is given on the techniques available and requirements for PCR based on minute templates. Different pre-PCR approaches are briefly described and possibilities and limitations of using archival material compared to fresh frozen tissue are discussed. In the article we give one example on how we have used the PALM laser microscopy system in combination with a nested, multiplex PCR system to analyze single normal keratinocytes as well as tumor cells from a case of basal cell cancer. We found that p53 mutations are common in normal, chronically sun-exposed skin. Widespread yet common mutations in the p53 gene that were unrelated to immunoreactivity for the p53 antibody were found in tumor cells. In addition there were rare mutations in occasional tumor cells that apparently did not result in selective growth advantage. Perspectives for the future are presented and the potential of laser assisted microdissection is highlighted within the fields of cancer research, developmental studies as well as studies of inflammatory and degenerative diseases. The combination of a method that allows careful selection of defined cells with powerful micro array based techniques, provides a setting with potential to uncover pathogenic mechanisms for large variety of human diseases.

Cytological Techniques↗

PubMed2004

[Detection of minimal residual disease of chronic myeloid leukemia patients after allogeneic stem cell transplantation by combination of STR-PCR with RT-PCR].

In order to evaluate relapse predication ability of STR-PCR combining with qualitative RT-PCR for the bar/abl transcripts to the patient with chronic myeloid leukemia (CML) fulfilled allogeneic stem cell transplantation (allo-HSCT), 24 patients with CML after allo-HSCT were dynamically investigated for MRD, quantitative analysis of donor chimerism was performed by multiplex PCR amplification of STR markers and capillary electrophoresis with fluorescence detection, qualitative detection of bcr/abl transcripts was detected by nested RT-PCR. The results showed that persistent full donor chimerism (DC >/= 95%) was associated with an absence of MRD. All patients with stable MC (90% </= DC < 95%) and bcr/abl negative had a probability of long-term survival with molecular remission, however the result of bcr/abl positivity was not always associated with leukemia relapse, only the patient with decreasing values of donor chimerism as well as bcr/abl positive proved to be in a higher risk of relapse or graft failure. Decrease of donor chimerism in correlation with MRD positive was detected in 5 patients. Three out of five patients had been proved to have a molecular relapse, one out of five patients had developed to cytogenetic relapse and another patient experienced graft failure. It is concluded that the results of STR-PCR in the range of its sensitivity fully correspond with bcr/abl tests in patients. The combination of STR-PCR with RT-PCR will provide a highly sensitive and valuable tool for MRD detection in CML and can identify these high risk patients with molecular or cytogenetic relapse after allo-HSCT.

Genes, abl↗