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At least 649 records · Page 36Linked to original sources
PubMed2003

Clinical screening of gene rearrangements in childhood leukemia by using a multiplex polymerase chain reaction-microarray approach.

PURPOSE: Currently, many forms of leukemia are considered potentially curable, with prognosis and clinical outcome strongly dependent on the underlying molecular pathophysiology. A substantial number of leukemia patients harbor nonrandom karyotypic abnormalities that define subgroups with unique biological and clinical features. For detection of these types of gene rearrangements, a combination of multiplex RT-PCR with hybridization on oligonucleotide gel array was presented previously, which identified five chromosomal translocations with fusion variants. In the present study, additional clinically relevant translocations were included in our analysis using a second generation of microarrays. We also expanded significantly on the clinical correlation of our findings. EXPERIMENTAL DESIGN: An oligonucleotide microarray was designed for hybridization with products of a multiplex RT-PCR to identify the following translocations: t(9;22)p190, t(4;11), t(12;21), t(1;19), typical for acute lymphoblastic leukemia; t(9;22)p210 for chronic myeloid leukemia; and t(8;21), t(15;17), inv16, typical for acute myeloblastic leukemia. RESULTS: To demonstrate the potential clinical application of the method, 247 cases of childhood leukemia were screened, and the above-mentioned gene rearrangements were found in 30% of cases. The sensitivity and specificity of the assay is comparable with the RT-PCR technique, so that it can be used to follow minimal residual disease. The feasibility of an additional refinement of the method, on-chip-multiplex PCR, has been successfully demonstrated by identifying a common translocation, t(9;22), in chronic myeloid leukemia. CONCLUSIONS: Our data suggest that the microarray-based assay can be an effective and reliable tool in the clinical screening of leukemia patients for the presence of specific gene rearrangements with important diagnostic and prognostic implications. The method is amenable for automation and high-throughput analysis.

Alternative Splicing↗
PubMed2003

[Distributions of haplotypes for three Y-STR loci in a Tibetan ethnic group of Chinese population by using Y-STR multiplexes].

OBJECTIVE: One multiplex genotyping system was developed in using silver staining with allelic ladders for three Y-chromosome STR markers (DYS434, DYS443, and DYS456), with a view towards the application of rapid and simple genotyping assay methods for DNA profiling. The distributions of haplotypes for three Y-STR loci(DYS434, DYS443, and DYS456) was investigated in a Tibetan ethnic group of Chinese population. METHODS: Allele and haplotype frequencies at these Y-STRs loci(DYS434, DYS443, and DYS456) were analysed by PCR amplification using Y-STR multiplexes, followed by horizontal non-denaturing polyacrylamide gelelec-trophoresis in 101 unrelated males of Tibetan ethnic group in Lasa of China. RESULTS: A total of 31 different haplotypes were found, 16 of them being unique. The haplotype diversity value (which is the same as the discrimination index) calculated from all three loci combined was 0.9481, which is informative. CONCLUSION: The Y-STR multiplexes provide useful information for forensic analysis and paternity tests and can also be of great benefit for providing information not normally available from autosomal DNA systems.

Alleles↗
PubMed2003

Validation of a multiplex RT-PCR assay for screening significant oncogene fusion transcripts in children with acute lymphoblastic leukaemia.

In childhood acute lymphoblastic leukaemia (ALL), cytogenetics play an important role in diagnosis, allocation of treatment and prognosis. Conventional cytogenetic analysis, involving mainly karyotyping in our experience, has not been successful in a large proportion of cases due to inadequate metaphase spreads and poor chromosome morphology. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent fusion transcripts resulting from specific chromosomal translocations, namely, both the CML- and ALLtype BCR-ABL transcripts of t(9;22), E2A-PBX1 transcript of t(1;19), the MLL-AF4 transcript of t(4;11) and TEL-AML1 (also termed ETV6-CBFA2) of the cryptic t(12;21). A multiplex reverse transcription polymerase chain reaction protocol (RT-PCR) was developed and tested out on archival bone marrow samples and leukaemia cell lines. In all samples with a known translocation detected by cytogenetic techniques, the same translocation was identified by the multiplex-PCR assay. Multiplex RT-PCR assay is an effective, sensitive, accurate and cost-effective diagnostic tool which can improve our ability to accurately and rapidly risk-stratify patients with childhood ALL.

Child↗
PubMed2004

Rapid detection of bacterial atypical pneumonia agents by multiplex PCR.

Approximately one third of community acquired pneumonia cases are caused by atypical pneumonia agents, Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydophila pneumoniae (formerly Chlamydia pneumoniae). The laboratory diagnosis of these organisms is difficult and time-consuming by conventional microbiological techniques. Polymerase chain reaction (PCR) is one of the important tools which can circumvent this problem. A multiplex PCR assay was developed to achieve the diagnosis of these three organisms in a single tube. Primers used in PCR were selected in a way that they amplified different length DNA fragments from different agents but they all worked at the same amplification conditions. Therefore the organisms could be diagnosed according to the length of amplified products by agarose gel electrophoresis without using any hybridization probes. After development of the multiplex PCR method, totally 309 clinical samples which were sent to our laboratory for single-agent PCR, were also evaluated by this technique. The results showed that the multiplex PCR assay is a sensitive, useful, cheap, and rapid diagnostic tool for the management of pneumonia patients.

Chlamydophila pneumoniae↗
PubMed2004

[Polymorphism and multiplex amplification of 3 X-chromosome specific short tandem repeat loci].

OBJECTIVE: To devise a multiplex PCR system of three X-chromosome specific short tandem repeat (X-STR) loci and study the genetic polymorphism. METHODS: DXS6799, DXS6804 and DXS6854 were amplified simultaneously using a multiplex system and were typed by polyacrylamide gel electrophoresis and silver staining. RESULTS: A total of 262 male and 255 female individuals from Guangdong Han population were tested; each locus showed 7 alleles. 73 haplotypes were detected in the male individuals. The haplotype diversity reached 0.9674. CONCLUSION: The 3 X-STR multiplex system is relatively abundant in polymorphic information for forensic identification and paternity testing.

Chromosome Mapping↗
PubMed2004

[Scanning HNF-1 alpha gene mutation in Chinese early-onset and/or multiplex diabetes pedigrees].

OBJECTIVE: To investigate the prevalence of mutations of hepatocyte nuclear factor (HNF)-1 alpha gene in Chinese families with early-onset and/or multiplex diabetes mellitus. METHODS: The studied population consisted of 247 unrelated Chinese residents in Shanghai, including 93 healthy controls and 154 probands of early-onset and/or multiplex diabetes pedigrees. The ten exons, flanking introns and minimal promoter region of HNF-1 alpha gene were screened using polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. RESULTS: Fourteen substitutions were identified in 154 probands. Three variants were not observed in 93 healthy controls. Two of them (nt-128T-->G IVS2 nt+21G-->A) were not reported previously and all co-segregated with diabetes. The genotype and allele frequencies of the other eleven variants in the diabetic patients were not significantly different from those in the healthy controls. There were no significant relationships between the eleven variants of HNF-1 alpha gene and clinical variables (plasma glucose, insulin, C-peptide and fasting lipid profile). CONCLUSION: HNF-1 alpha gene is not a major cause of early-onset or multiplex diabetes pedigrees in this Chinese population in Shanghai.

Asian People↗
PubMed2004

[Ilizarov technique for correcting flexion deformity of the knee of arthrogryposis multiplex congenita].

OBJECTIVE: To study the methods and effects of Ilizarov distraction technique in treating the flexion deformity of the knee of arthrogryposis multiplex congenita. METHODS: Between August 1998 and February 2003, 6 patients (10 knees) with the arthrogryposis multiplex congenita were treated, 4 patients in double knees, 5 males, 1 female, mean age 8 years and 2 months, ranged from 3 years and 7 months to 13 years. The preoperative flexion degree was averagely 51 degrees. The patients accompanied 13 other parts malformation of limbs. The modified Ilizarov distraction apparatus of the knee was used. While installing the apparatus in the operation, the knees should be kept in the of location of maximum extension, the center of joint hinges on the apparatus should be placed towards the rotatory center of the knee, two groups of 2 mm K-wires were passed through the femur and tibia around the knee, who were fixed on the proximal and distal rings. Distraction was started after the surgical procedure 5 days via rotating the threaded rods at the posterior of the knee, at an average of 2 to 3 mm per day, at the first week, after 2 weeks with the rate modified to 2 mm per day, up to the knee extended to 0 degrees. The accompanied deformities of the hip and/or the foot might be corrected at the same time or next time. The average duration of the distraction was 37 days (23-48 days). During the correction all limbs might undergo weight. After 2 weeks at the end of distraction the fixator was removed and the patients could walk by a long-leg brace. RESULTS: Ten knees with the flexion contracture were sufficiently corrected without severe complications. Nine knees of all were followed up at an average time of 1 years and 3 mouths, no recurrence of the deformity was seen in all patients, their function of walk was significantly improved. CONCLUSIONS: Ilizarov technique is a simply, safe and effective method for managing the flexion deformity of the knee of the arthrogryposis multiplex congentia. The procedure is conformable to the biological theories and microsurgical principles.

Adolescent↗
PubMed2003

[Four Y-STR multiplex system by silver staining].

A multiplex PCR system has been developed to amplify 4 Y-chromosome specific short tandem repeats (STR), DYS391, GATA-A4, GATA-A10 and GATA-H4, simultaneously. PCR products were separated by polyacrylamide gels electrophoresis followed by silver stain. When 311 unrelated males from the Han population in Guangdong were detected by the multiplex system, DYS391, GATA-A4, GATA-A10 and GATA-H4 showed 5, 7, 6 and 5 alleles respectively. Total 98 haplotypes could be identified. Gene diversity value for the 4 STR was 0.4623, 0.6972, 0.7173 and 0.6015 respectively. The gene diversity value for the haplotypes of the 4 Y-STR reached 0.9755. The four Y-STR multiplex system will be very powerful for establishing Y-STR database, exploring human origin, paternity testing and personal identification.

English Abstract↗
PubMed2005

Development and application of a multiplex PCR procedure for the detection of DNA methylation in colorectal cancer.

Cancers often exhibit aberrant methylation of gene promoter regions associated with loss of tumor suppressor and/or DNA repair gene function. Such methylation constitutes an excellent marker for the molecular detection of micro-metastases and the diagnosis of tumor recurrences. We have developed a multiplex methylation-specific PCR (MSP) procedure for rapid and simultaneous assessment of the methylation of 5 loci: the tumor suppressor genes p16INK4a, death-associated protein kinase (DAPK) and p14ARF, and the DNA repair genes hMLH1 and O6-methylguanine-DNA-methyltransferase (MGMT). This multiplex test uses one single PCR reaction and only one electrophoretic run. In 98 samples of colorectal cancer studied, methylation of MGMT, DAPK, p16, hMLH1 and p14 was present in 31, 20, 17, 16 and 14% of tumors, respectively. In 58% of the tumors at least one methylated gene was found. This multiplex MSP constitutes a simple and inexpensive method for screening of molecular signatures in colorectal cancer and can be used profitably before employing more expensive and complex techniques such as microarray testing.

Adaptor Proteins, Signal Transducing↗
PubMed2004

[Comparison of effect between multiplex and simplex nutritional intervention on hyperlipemia, hypertension or diabetes].

OBJECTIVE: To explore the effect of simplex and multiplex nutritional intervention on hyperlipemia, hypertension or diabetes. METHODS: 260 persons who have hyperlipemia were randomized to 3 groups: (1) "the multiplex nutritional intervention group", is given 100 g coarse food grain everyday and nutrition education, (2) "the simplex nutritional intervention group", is only given nutrition education, (3) "the control group". RESULTS: After three months, the degressive value of blood pressure, blood sugar and blood lipid respectively in the multiplex nutritional intervention group were larger than in the simplex nutritional intervention. CONCLUSION: There are better effect on hyperlipemia, hypertension and diabetes by nutrition education plus passive dietary intervention than by nutrition education.

Aged↗
PubMed2005

[Detecting haplotypes of four Y-STR by multiplex amplification].

OBJECTIVE: To establish a multiplexing Y-STR system and study haplotype frequencies of 4 Y-specific loci in China Han population. METHODS: DYS439, DYS390, GATA-A7.2 and DYS393 loci were amplified simultaneously and were analyzed by polyacrylamide gel electrophoresis and silver staining. RESULTS: When 558 unrelated male individuals from the Han population in China were tested by the multiplex system, DYS439, DYS390, GATA-A7.2 and DYS393 show 7,7,7,6 alleles, respectively. 180 different haplotypes were detected. The power of discrimination of this system was 0.9853. CONCLUSION: The multiplex amplified system of these 4 Y-specific loci and their database are useful for human origin exploration and forensic practice.

Alleles↗
PubMed2005

[The STR typing system by fluorescence labeled Multiplex-PCR technique and its forensic application].

OBJECTIVE: To build the four STR loci typing system by fluorescence labeled Multiplex-PCR technique, applied in the parentage test and personal identification in forensic medicine. METHODS: The primer of D3S1754 and D1S549 were labeled with 6-FAM and TMR respectively, primers of D4S2366 and D12S375 were labeled with HEX. Multiplex-PCR products were analysed on the ABI PRISM 310 Genetic Analyzer where the Data Collection Software 3.0, the GeneScan Analysis Software 3.7NT and the Genotyper 3.7NT Software were used. This typing system has been emploied in the parentage test and personal identification of casework. RESULTS: A method of typing four STR loci by fluorescence labeled Multiplex-PCR technique had been constructed. It has showed good sensitive and stability, and met the needs of parentage test and personal identification in forensic medicine. CONCLUSION: The constructed method can be used in studying genetic polymorphisms and parentage test or personal identification in forensic medicine.

DNA↗
PubMed2005

DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach.

Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.

Base Sequence↗
PubMed2005

Optimization of a multiplex minisequencing protocol for population studies and medical genetics.

Several technologically sophisticated high-throughput techniques have been recently developed for the study of human single nucleotide polymorphisms and the diagnosis of point mutations in human diseases. However, there is also a need for simple and inexpensive techniques suitable for clinical services and small research laboratories. Minisequencing meets the latter requirements. It is simple, non-radioactive and can be easily multiplexed by adding oligonucleotide tails of increasing size to the sequencing oligonucleotide primers. To optimize the minisequencing protocol, we designed a test multiplex system capable of typing simultaneously 12 different human autosomal single nucleotide polymorphisms. We discovered that the quality of minisequencing primers and the careful selection of the tail sequences were especially critical for success. This optimized protocol permits rapid genotyping at low cost and can serve as a blueprint for the creation of multiplex minisequencing systems suitable to virtually any typing application in population studies and medical genetics.

DNA Primers↗
PubMed2005

[Detection of virulence-associated factors of Streptococcus suis by multiplex PCR assay].

OBJECTIVE: To rapidly and sensitively detect the four virulence-associated factors of Streptococcus suis, a multiplex PCR was developed. METHODS: In the process of this reaction, four distinct DNA targets were amplified. One target was based on the serotype 2 (and 1/2) specific cps gene and the others were based on Streptococcus suis mrp, epf (epf*) and sly gene, encoding the MRP, EF(EF*) and Sly proteins of Streptococcus suis. 72 isolates, which including 48 strains of Streptococcus suis and 24 strains of negative control, and 49 clinical specimens were detected by the multiplex PCR assay. RESULTS: All PCR products were detected by electrophoresis on 1.2% agarose gels. With the 48 Streptococcus suis strains, the positive detection rates of cps2+, mrp+, epf+, epf*+ and sly+ were 16/48, 14/48, 12/48, 3/48 and 26/48,respectively. The results were confirmed by bacteriological examination. There were no specific amplification products including 49 clinical specimens and 24 negative control strains. CONCLUSION: The results demonstrated that multiplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of streptococcus suis.

Bacterial Proteins↗
PubMed2005

[Development and application of multiplex-PCR for identification of Actinobacillus pleuropneumoniae].

A multiplex-PCR assay was developed to identify Actinobacillus pleuropneumoniae (App). Two pairs of polymerase chain reaction (PCR) primers were designed for the 16S rRNA and the apxlVA gene, which is specific to all serotypes of App. Two PCR products of 692bp and 363bp were obtained, from the 16S rRNA and the apxlVA gene respectively, for 27 reference A. pleuropneumoniae strains. Only the 692bp fragment was amplified for closely related strains including A. lignieresii. Using the designed primers, the method is capable of detecting A. pleuropneumoniae of as low as 1.3 x 10(3) CFU or 9pg DNA. For 302 suspected isolates, this multiplex-PCR method correctly identified 4 A. pleuropneumoniae strains. The result suggests the use of the multiplex-PCR for routine identification of App.

Actinobacillus pleuropneumoniae↗
PubMed2005

[Primarily study on a multiplex PCR-based system for the rapid detection of Salmonella spp., Shigella spp. and Escherichia coli O157:H7].

OBJECTIVE: To establish a multiplex PCR-based system for the simultaneous detection of Salmonella spp., Shigella spp. and Escherichia coli 0157:H7 in 12 hours. METHODS: After 6 h nonselective enrichment in BPW, DNA template were prepared at 100 degrees C for 10 min. Three sets of primers were designed to amplify the gene segments of invA of Salmonella spp, ipaH of Shigella spp, and uidA of E. coli 0157:H7, and the products were analyzed by electrophoresis. At the same time, this system was optimized, and the specificity and sensitivity of this system were evaluated. RESULTS: Three target bacteria were detected in 12 h by using this multiplex PCR-based system. The sensitivity of it was up to 10-30 cfu/ml, and the high specificity was demonstrated by detecting 23 target stains and 15 non-target stains. CONCLUSION: A rapid, specific, and sensitive multiplex PCR-based system for the simultaneous detection of Salmonella spp., Shigella spp. and E. coli 0157:H7 in 12 h has been studied primarily.

Escherichia coli O157↗
PubMed2005

[Utility of multiplex PCR for diagnosis of neurological disorders in HIV-infected patients and patients with AIDS].

OBJECTIVE: Evaluation of multiplex PCR utility for diagnosis of central nervous system disorders in HIV-infected patients. METHODS: Multiplex PCR assay for simultaneous detection of HSV-1, HSV-2, VZV, HHV-6, CMV, EBV, JCV, T. gondii DNA in 27 CSF samples from HIV-infected patients was used. PCR results were compared with clinical diagnosis and histopathological examination of brain obtained at autopsy. MAIN OBSERVATIONS: Five of seven positive results were corresponding with clinical diagnosis. In two cases also with autopsy. RESULTS: DNA of one or more pathogens was found in 6 out of 27 (22%) samples: CMV DNA in four patients, JCV DNA and T. gondii DNA in one patients each. In one case co-infections of CMV and EBV was found. CONCLUSIONS: Multiplex PCR is a useful diagnostic method. LIPA-CNS test has high specificity. Lack of nesting step of the reaction and PCR positive control verification could cause a relatively low sensitivity of the test.

AIDS-Related Opportunistic Infections↗