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At least 325 records · Page 18Linked to original sources
PubMed2003

Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome.

Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.

Animals↗
PubMed2003

[Multiplex PCR normalization and parallel detection of HBV and HCV].

BACKGROUND: To design and establish a method of multiplex PCR normalization and parallel detection of HBV and HCV. METHODS: Using two pairs of primers, one inner and the other outer, each having a 20 bp common sequence, the authors amplified target DNA for two rounds. All products would have this common sequence. Using this common sequence as primer the authors performed further amplification. Finally, multiplex PCR was normalized to a single PCR style and eliminated multiple factors disturbance. Four kinds of nucleic acid extraction method were compared. Multiplex PCR normalization was established and optimized by using orthogonal design by analysing 6 kinds of key factors. The method was evaluated by detecting 28 samples of HBV and HCV. RESULTS: The sensitivity, specificity, diagnostic idex and efficiency for HBsAg, HCV antibody positive patients were 83.3%, 70.0%, 153.3%, 72.2% respectively. The sensitivity, specificity, idex and efficiency for HBsAg positive patients were 78.6%, 80.0%, 158.6%, and 79.2%, respectively. The sensitivity, specificity, idex and efficiency for HCV antibody positive patients were 75.0%, 90.0%, 165.0% and 83.3% respectively. CONCLUSIONS: The multiplex PCR normalization method may have potential applicability in parallel amplification of multiple genes of pathogens.

DNA, Viral↗
PubMed1992

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.

Base Sequence↗
PubMed2003

Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar typhimurium.

A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.

Polymerase Chain Reaction↗
PubMed2003

Multiplex PCR identification of wheat HMW glutenin subunit genes by allele-specific markers.

Wheat bread-making quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular weight (HMW) glutenin subunits encoded by the Glu-1 genes. A multiplex polymerase chain reaction (PCR) method was developed to identify the allele composition of HMW glutenin complex Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) in common wheat genotypes. The study of multiplex PCR to obtain a well-balanced set of amplicons involved examination of various combinations of selected primer sets and/or thermal cycling conditions. One to three simultaneously amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis and differences between Ax1, Ax2* and Axnull genes of Glu-A1 loci, Bx6, Bx7 and Bx17 of Glu-B1, and Dx2, Dx5 and Dy10 genes of Glu-D1 loci were revealed. A complete agreement was found in identification of HMW glutenin subunits by both multiplex PCR analysis and SDS-PAGE for seventy-six Polish cultivars/strains of both spring and winter common wheat. Rapid identification of molecular markers of Glu-1 alleles by multiplex PCR can be an efficient alternative to the standard separation procedure for early selection of useful wheat genotypes with good bread-making quality.

Alleles↗
PubMed2003

[Large deletion in mismatch repair genes uncovered by quantitative multiplex PCR-high performance liquid chromatography system].

OBJECTIVE: Establishing a new method on the basis of multiplex PCR-high performance liquid chromatography (HPLC) for screening a large deletion in mismatch repair genes. METHODS: Thirty-five pairs of primers were used to amplify all 16 exons of MSH2 and all 19 exons of MLH1 gene in 8 multiplex PCR. The products of multiplex PCR were analysed for the large deletion with Double Strand DNA Analysis System of HPLC. Firstly, validation of the method was tested on positive and negative controls in blind analysis. Secondly, 14 blood cell DNA samples from hereditary nonpolyposis colorectal cancer (HNPCC) patients and 13 colorectal cancer (CRC) tissues DNA samples from sporadic CRC patients were checked with the new developed method. RESULTS: (1) the genomic deletions in all 4 of positive controls were identically uncovered with the new method; (2) a novel germline and a novel somatic large deletions were unveiled in 1/14 HNPCC patients and in 1/13 CRC tissues. CONCLUSION: The method developed on multiplex PCR-HPLC is reliable for uncovering large genomic deletion in mismatch repair genes, and can be taken as a valuable addition to mutation screening system.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Real-time rectilinear 3-D ultrasound using receive mode multiplexing.

In previous work, we developed two generations of a real-time rectilinear volumetric scanner operating at 5 MHz for abdominal, breast, or vascular imaging using a Mills cross two-dimensional (2-D) array and a rectilinear periodic 2-D array. To improve spatial resolution performance and sensitivity, we developed a new design using 4:1 receive mode multiplexing. With 4:1 multiplexing, the new 65,000 element 2-D array has 4 x 256 = 1024 receivers so that 256 receivers can be used on any image line. The two major benefits of using receive mode multiplexing are an increase in receive sensitivity due to a greater number of receive elements, and a decrease in grating lobe and clutter levels due to increased receive element density. Theoretical simulations and analysis show an increase of about 13 dB in sensitivity compared to our previous work. With these encouraging results, a new 65,000 element 5-MHz, 2-D array having 1024 receivers and 169 transmitters was prototyped. In addition, the multiplexer and control circuitry were designed, built, and interfaced with both the transducer and volumetric scanner. Images of tissue-mimicking phantoms and in vivo targets were obtained. Using a spherical cyst phantom, experimental results showed a +12 dB improvement in signal-to-noise ratio and a +6 dB improvement in contrast compared to our previous work.

Adult↗
PubMed2004

[Amplification of pig microsatellite markers using multiplex PCR].

In order to rapidly amplify pig microsatellite markers and save materials,multiplex PCR was used and its reaction condition was optimized. Forty-six combinations of multiplex PCR with good effects were obtained. Thirty of them are duplex-PCRs, sixteen are triplex-PCRs. The results of multiplexes showed that the concentration of primers varied among 0.06 approximately 0.3 micromol/L, the Mg(2+) concentration among 1.5 approximately 3.0 mmol/L; 0.2 approximately 0.4 U of Taq polymerase and 1.0-, 1.2-, 1.4-, 1.6-fold buffer were used, the annealing temperature and the cycle number varied among 52 approximately 60 degrees and 32 approximately 50 degrees, respectively. All multiplexes were further combined into 17 sets for the electrophoresis on ABI 377 sequencer.

Animals↗
PubMed2005

[Distribution of haplotypes for four Y-sTR loci and validation in forensic science by using a double-fluorescent multiplex PCR system].

OBJECTIVE: We focus on developing a multiplex PCR system for Y-STR loci that can be detected by double fluorescent system and assessing their usefulness in forensic mixture samples. METHODS: The primers of four Y-STR loci (DYS-GATA-A10, DYS531, DYS557 and DYS448) amplified by multiplex PCR technique were labeled with fluorescence, then the PCR products of these Y-STRs loci were detecting and typing by ABI PRISM310 Genetic Analyzer. RESULTS: When 120 unrelated individuals from the Han population in Chengdu were detected by the system, Y-GATA-A10, DYS531, DYS557 and DYS448 showed 5, 5, 8, 7 alleles, respectively. A total of 78 different haplotypes was identified and the genetic diversity reached 0.9881. To the three cases of mixture stains failed by using conventional autosomal STR analysis, our multiplex system drew conforming conclusion comparing to the suspect's Y-STRs genotypes. CONCLUSION: Our results show that the multiplex system of four Y-STR will be very powerful for Y-STR database establishing, the paternity testing and mixture stains identifying.

Alleles↗
PubMed2005

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water.

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.

Base Sequence↗
PubMed2005

[Development of multiplex reverse translation-polymerase chain reaction methods for detection of dengue virus type 1-4 and its application in clinical use].

OBJECTIVE: To develop multiplex reverse translation-polymerase chain reaction (RT-PCR) method for detection of dengue virus type 1-4. METHODS: Based on the genomes sequence analysis of dengue virus type 1-4, four-pair of primers were designed. The specificity of the primers was primarily tested by searching the GenBank DNA sequence database. The optimal reaction conditions of the multiplex RT-PCR were then established. The specificity of RT-PCR was tested using the homologous yellow fever virus and Japanese encephalitis virus. 30 serum samples of dengue virus from suspected sufferers in the prevalence of dengue virus in 2003 were detected using the methods we developed. RESULTS: Positive segments about 295, 237, 118, 347 bp could be seen in the multiplex RT-PCR production of dengue virus type 1-4, respectively. There were no positive segments in the RT-PCR productions of Japanese encephalitis virus and yellow fever virus. 25 of the 30 serum samples showed dengue virus type 1 positive results, while the sequencing results suggesting the amplification sequence having a high homology with dengue virus type 1 strain Cambodia, GD14/97 and GD05/99 (97%, 97%, 98%, respective). CONCLUSION: The method of multiplex RT-PCR we established could be used for early detection and identification of dengue virus type 1-4.

Base Sequence↗
PubMed2005

[Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction].

OBJECTIVE: To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. METHODS: Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed. RESULTS: 200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30). CONCLUSION: PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.

Adult↗
PubMed2005

[Application of reverse transcription-multiplex nested PCR to detect MLL rearrangement in AML-M4/M5].

OBJECTIVE: To explore the value of reverse transcription-multiplex nested PCR in detecting MLL rearrangement in lzAML-M4/M5. METHODS: Bone marrow chromosome preparation was made using direct method or short-term culture. Karyotypic analysis was carried out by R-banding technique. Five common MLL fusion genes and MLL partial tandem duplication in 40 AML cases, including 12 M4 and 28 M5 were detected by reverse transcription(RT)-multiplex nested PCR. RESULTS: R-banding karyotypic analysis revealed 11q23 translocation including t(6;11)(q27;q23), t(9;11)(p21;q23), t(11;17)(q23;q21) and t(11;19)(q23;p13.1) in 7 cases. MLL rearrangements consisting of MLL/AF6 (1 case), MLL/AF9 (1 case), MLL/AF17 (2 cases), MLL/ELL (2 cases) and MLL partial tandem duplication(2 cases) were detected in 8 cases by RT-multiplex nested PCR. Among 8 cases with MLL rearrangement, 6 were chromosome translocation, 2 were MLL partial tandem duplication. CONCLUSION: RT-multiplex nested PCR is a powerful technique in the detection of MLL rearrangement for tentativelly diagnosed AML-M4/M5.

Adolescent↗
PubMed2005

Development of a novel protocol for RT-multiplex PCR to detect diarrheal viruses among infants and children with acute gastroenteritis in Eastern Russia.

Viral gastroenteritis is one of the most common illnesses in humans worldwide and it has a great impact on people. Recently, we reported three RT-multiplex PCR assays termed A, B and C that could detect three groups of diarrheal viruses; group A, B and C rotaviruses and adenovirus [Phan et al., J Med Virol 2004; 74:173-9]; norovirus GI and GII, sapovirus and astrovirus [Yan et al., J Virol Meth 2003; 114:37-44]; enteroviruses, hepatitis A and E viruses and influenza A virus [Phan et al., Arch Virol 2005; 1175-85], respectively. In the present study, we developed a novel protocol with a small volume of reaction mixture for RT and PCR products (only 8 microl and 11 microl, respectively) that can amplify genomes of target viruses simultaneously. A total of 100 fecal specimens from infants and children with acute gastroenteritis in Birobiclzhan city, Eastern Russia, were collected during November 2003 and March 2004 and tested for the presence of those viruses by the novel RT-multiplex PCR protocol. Group A rotavirus was the most prevalent (67%), followed by norovirus GII (4%), group C rotavirus (1%), and sapovirus (1%). Interestingly, one fecal specimen turned out to be positive for hepatitis A virus. The sensitivity and specificity of RT-multiplex PCR assays with a novel protocol demonstrated a strong validation against the previously published RT-multiplex PCR. The findings clearly indicated that this novel protocol is simple and cost-effective to investigate the molecular epidemiology of acute gastroenteritis caused by diarrheal viruses. This report is the first, to our knowledge, detecting hepatitis A virus in feces from diarrheal infants and children in Eastern Russia.

Child↗
PubMed2006

[Screening for Y chromosomal microdeletions in AZF region with modified multiplex PCR].

OBJECTIVE: Screening for Y chromosomal microdeletions in azoospermia factor (AZF) region with modified multiplex PCR. METHODS: 160 cases with spermatogenetic failure were recruited in the experimental group, while 90 cases of donors in controls. According to the laboratory guidelines supported by European Academy of Andrology (EAA) and European Molecular Genetics Quality Network (EMQN), Y chromosomal microdeletions in AZFa, b, c regions were screened with multiplex PCR. The primers of sequence targeted sites (STSs) and conditions of PCR were modified. RESULTS: Using modified multiplex PCR, 14 (8.75%) cases with Y chromosomal microdeletions were found in the experimental group, while no case in controls. There were 12 cases in AZFc, 1 case in AZFa + b + c, 1 case in AZFb + c. According to statistics, the difference between two groups was significant (P <0.001). Reaction products could be clearly separated with agarose gel and finished in 1 h. CONCLUSION: Modified multiplex PCR protocols supported by EAA and EMQN proved to be very accurate, sensitive and quick, which could be put into screening practice for Y chromosomal microdeletions in AZF region.

Adult↗
PubMed1996

Multiplex amplification and typing procedure for the loci D1S80 and amelogenin.

A method has been developed that enables multiplex amplification and simultaneous typing of the loci D1S80 and amelogenin using discontinuous polyacrylamide gel electrophoresis and silver staining. The protocol is sensitive, simple, rapid, and relatively inexpensive. The results of the multiplex analysis of the D1S80 and amelogenin loci were comparable to those obtained when each locus was analyzed individually. A small validation study was undertaken to evaluate the forensic applicability of this multiplex system. The data demonstrate that DNA exposed to a variety of environmental insults yields reliable multiplex typing results.

Amelogenin↗
PubMed1998

Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia.

We have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) reaction, which enables us to detect 29 translocations/chromosomal aberrations in patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). Through the construction and optimization of specific primers for each translocation, we have been able to reduce the set-up to 8 parallel multiplex PCR reactions, thus greatly decreasing the amount of work and reagents. We show the value of our set-up in a retrospective analysis on cryopreserved material from 102 AML and 62 ALL patients. The multiplex RT-PCR detected a hybrid mRNA resulting from a structural chromosomal aberration in 45 of 102 (44%) of the AML and in 28 of 62 (45%) of the pediatric ALL cases. Importantly, in 33% of AML and in 47% of the ALL cases with cytogenetic data, submicroscopic chromosomal aberrations or masked translocations were shown that were not detected in the cytogenetic analysis either for structural reasons or because of an insufficient number of metaphases obtained. This multiplex RT-PCR system, which can handle up to 10 patients with a response time of 2 working days, is thus an important tool that complements cytogenetic analysis in the up-front screening of acute leukemia patients and should provide a rapid and efficient characterization of leukemia cells, even in situations with sparse patient material.

Acute Disease↗
PubMed2018

Quantitation of DNA Methylation by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) Assay.

The defining feature of the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) method to sensitively quantify DNA methylation is the two-step PCR approach for a multiplexed analysis of a panel of up to 12 genes in clinical samples with minimal quantities of DNA. In the first step, for up to 12 genes tested, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction. This methylation-independent amplification step produces amplicons of up to 109 copies per &#x3bc;L after 36&#xa0;cycles of PCR. In the second step, the amplicons of the first reaction (STEP 1) are quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC). One methylated copy is detectable in 100,000 reference gene copies. Methylation is reported on a continuous scale. For the gene panel, the highest level of normal DNA methylation above which a sample would be called positive is derived by using Receiver Operating Characteristic (ROC), maximizing assay specificity and sensitivity to distinguish between normal/benign versus tumor DNA. QM-MSP can be applied to clinical samples of fresh or fixed ductal cells, ductal fluid, nipple fluid, fine needle aspirates, core biopsies, and tumor tissue sections.

Breast Neoplasms↗