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At least 415 records · Page 23Linked to original sources
PubMed2005

Differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction.

The objective of the present study was to develop a multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis coronavirus (IBV), and bovine coronavirus (BCoV). Primers were designed from conserved or variable regions of nucleocapsid (N) or spike (S) protein gene among TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by the PCR reaction was used to amplify a portion of N or S gene of the corresponding coronaviruses. The PCR products were detected on agarose gel stained with ethidium bromide. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, were obtained for TCoV isolates. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene was obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene was obtained for BCoV. There were no PCR products with the same primers for Newcastle disease virus, Marek's disease virus, turkey pox virus, pigeon pox virus, fowl pox virus, reovirus, infectious bursal disease virus, enterovirus, astrovirus, Salmonella enterica, Escherichia coli, and Mycoplasma gallisepticum. Performance of the assay with serially diluted RNA demonstrated that the multiplex PCR could detect 4.8x10(-3) microg of TCoV RNA, 4.6x10(-4) microg of IBV RNA, and 8.0x10(-2) microg of BCoV RNA. These results indicated that the multiplex PCR as established in the present study is a rapid, sensitive, and specific method for differential detection of TCoV, IBV, and BCoV in a single PCR reaction.

Animals↗
PubMed2005

Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection.

H5N1 influenza A virus causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to mammalian species such as leopards, tigers and humans. The aim of this study was to develop a multiplex real-time RT-PCR for rapid detection of H5N1 influenza A virus. The selected primers and various labeled TaqMan MGB reporter probes corresponding to M, H5 and N1 were used in a single step multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. In order to validate the method, 75 clinical specimens infected with H5N1 isolated from both poultry and mammals, as well as various specimens of other subtypes and RNA from other viral pathogens of poultry and human were tested. The results showed that the multiplex real-time RT-PCR assays can be applied to detect virus suspensions of H5N1 influenza A virus from a wide host range and demonstrated the sensitivity of the assay amounted to approximately 10(2)-10(3)copies/mul. In conclusion, the highlights of this particular method lie in its rapidity, specificity and sensitivity thus rendering it feasible and effective for large-scale screening at times of H5N1 influenza A virus outbreaks.

Animals↗
PubMed2004

The use of different multiplex PCRs for twin zygosity determination and its application in forensic trace analysis.

STR typing becomes more and more valuable for different approaches of science. It revolutionized determining of zygosity in twin research and it is very often the only possibility for discrimination in forensic trace analysis. In this study 55 twin pairs from Denmark were genetically investigated to determine their zygosity. For analysis two multiplex PCR kits, the AmpFlSTR Identifiler kit, which comprises 15 loci plus the amelogenin gender determination and the Powerplex ES kit with eight different loci were employed. For a forensic approach every twin pair was regarded as being a forensic trace analysis, i.e. suspect or victim/biological trace and then we determined the security and precision with that a match of genetic patterns or an exclusion could be observed. Sixty percent of the twin pairs were di- and 40% monozygotic. There were no differences in zygosity determination between the two multiplex kits. The lowest number of exclusions by determining dizygosity was four loci for the Identifiler and three for Powerplex ES kit, the highest was 13 (Identifiler) and eight (Powerplex). It could be shown that the highly discriminative multiplex PCR kits gave matching probabilities of over 99.999999% (calculation based on data for unrelated individuals) even when only five or six STRs could be determined (assuming a trace analysis with some non-detectable STRs and therefore an incomplete genetic pattern). No questionable results regarding zygosity of the twin pairs were obtained even when only eight loci (using the Powerplex ES kit) were investigated. The simulated forensic results showed that matching probabilities should always be handled with care to not possibly come to wrong conclusions concerning the origin of the biological trace.

Alleles↗
PubMed2004

Four-color multiplex 5' nuclease assay for the simultaneous detection of the factor V Leiden and the prothrombin G20210A mutations.

We developed a real-time multiplex four-color assay for the simultaneous detection of the factor V Leiden (FVL) and prothrombin (PT) G20210A mutations in one closed tube using a single thermocycling protocol. The assay combines the power of multiplex PCR with the specificity provided by allele-specific oligonucleotide (ASO) hybridization using the 5' nuclease assay format. Human genomic DNA is prepared from whole blood with standard procedures. A 97-bp DNA sequence of the coagulation factor V gene is co-amplified with a 111-bp DNA sequence of the coagulation factor II (PT) gene using four PCR primers. In addition, the reactions included four differentially labeled ASO probes for the specific detection of the different FVL/PT G20210A genotypes. To evaluate the assay's performance characteristics, we performed a comparison of two methods. We analyzed DNA samples from 52 individuals with known FVL/PT G20210A genotypes that were previously genotyped with an assay that combined PCR with the use of restriction fragment length polymorphisms. We found a 100% concordance between the results generated by both methodologies. We conclude that the four-color multiplex assay is specific and reproducible for the detection of the FVL/PT G20210A mutations, and it can be easily adapted for the detection of other SNPs.

Alleles↗
PubMed2005

Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR.

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, but more serious complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) can result. A real-time PCR method to detect the presence of Shiga toxin producing E. coli (STEC) and E. coli O157:H7 was investigated using SYBR Green I (SG). Primers were designed to target the Shiga toxin genes (stx1 and stx2) and a highly conserved base substitution at +93 of the beta-glucuronidase gene (uidA) unique to E. coli O157:H7. An initial test panel of five E. coli and non-E. coli isolates was tested with individual primer sets (simplex assay) and all primer sets including stx1, stx2, and uidA (multiplex assay). All strains were correctly identified in both assays. Average melt temperatures (Tm's, degrees C) for PCR products were 85.42--stx1, 81.93--stx2, and 88.25--uidA in simplex assays and 85.20--stx1, 81.20--stx2, and 88.16--uidA when multiplexed. Each of the three gene targets in one multiplex reaction could be distinguished by melt curve data with significantly different Tm's. The assay was expanded to a panel of 138 isolates consisting of STEC, E. coli O157:H7, non-toxigenic E. coli, and non-E. coli isolates with melt peaks consistent with those stated above.

Benzothiazoles↗
PubMed2005

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli.

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.

Adhesins, Bacterial↗
PubMed2005

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum.

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.

Animals↗
PubMed2006

Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes.

Escherichia coli 0157:H7 and Listeria monocytogenes are the two most important food-borne human pathogens. To develop a single, rapid and sensitive PCR based test for simultaneous detection of both the organisms, fliCh7 and iap gene specific primers were used respectively for E. coli 0157:H7 and L. monocytogenes. Initially, with equal quantities of purified genomic DNAs of these organisms a multiplex PCR reaction was standardized to yield uniform amplification of both targets. Although, this assay detected E. coli 0157:H7 with high sensitivity, it failed to pick up L. monocytogenes after several hours of enrichment in broth medium initially spiked with equal numbers of live cells. This was found to be due to unequal growth of these organisms leading to disparity in the amount of template DNAs represented in the DNA preparation applied for conventional multiplex PCR amplification. To circumvent this, we have developed a modified method of enrichment and harvesting leading to highly sensitive and rapid single reaction PCR detection of both pathogens. We have also successfully developed two novel multiplex PCR formats for the generation of uniform PCR signals. Some of these methods might find broader application for the simultaneous detection of different combinations of multiple pathogens.

Bacterial Proteins↗
PubMed2006

[Simultaneous detection by multiplex PCR of atypical bacterial pathogens involved in infections of respiratory tract. Is it useful for the microbiological diagnosis of respiratory infections].

The aim was to evaluate the "strategical place" of the new commercial test Chlamylege (Argene-Biosoft-France) which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae and most Legionella species using a PCR multiplex. 41 patients with an atypical pneumonia were included, all standard procedures of diagnosis were done and in addition the chamylege test. A pathogen was identified in 12 patients, an other microorganisms than the 3 targeted by our study was found in 8 patients. 4 positive PCR were obtained, 3 with M. pneumoniae and 1 with Legionella pneumophila 1. That means that for 29 patients no aetiology was found. Among them 23 clearly improved under antibiotic treatment. Though that PCR multiplex is an attractive test, easy to perform, sensitive, specific and convenient, we need further studies to approach the place of this PCR test in the diagnosis of multifaceted atypical pneumonia. We also need to know if the cost associated with the microbiological diagnosis (culture, serology, immunofluorescence, urinary antigen test, PCR...) for atypical pneumonia worth value? An algorithm as to be drawn to determine the value of intensive microbiological investigation. An other point to discuss, may be this kind of rapid and multiplex PCR technique could lead to spare the use of some antibiotics.

Atypical Bacterial Forms↗
PubMed2026

Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.

The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.

Solanum tuberosum↗
PubMed2005

Multiplex MQMAS NMR of quadrupolar nuclei.

A multiplex phase cycling method (N. Ivchenko et al., J. Magn. Reson. 160 (2003) 52-58) has been used to record two-dimensional MQMAS spectra with a very short phase cycling. A straightforward procedure has been developed to easily process the data. Combining this Multiplex approach and the new Soft-Pulse-Adding-Mixing (SPAM) method considerably increases the signal-to-noise ratio of the conventional MQMAS experiment. The Multiplex acquisition procedure is much simpler than the echo/anti-echo method recently proposed, and has been applied with success to record (87)Rb spectra of RbNO(3) and (27)Al 3Q and 5Q MQMAS NMR of microporous aluminophosphate AlPO(4)-14.

Journal Article↗
PubMed2005

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood.

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.

Animals↗
PubMed2024

Mudskipper detects combinatorial RNA binding protein interactions in multiplexed CLIP data.

The uncovering of protein-RNA interactions enables a deeper understanding of RNA processing. Recent multiplexed crosslinking and immunoprecipitation (CLIP) technologies such as antibody-barcoded eCLIP (ABC) dramatically increase the throughput of mapping RNA binding protein (RBP) binding sites. However, multiplex CLIP datasets are multivariate, and each RBP suffers non-uniform signal-to-noise ratio. To address this, we developed Mudskipper, a versatile computational suite comprising two components: a Dirichlet multinomial mixture model to account for the multivariate nature of ABC datasets and a softmasking approach that identifies and removes non-specific protein-RNA interactions in RBPs with low signal-to-noise ratio. Mudskipper demonstrates superior precision and recall over existing tools on multiplex datasets and supports analysis of repetitive elements and small non-coding RNAs. Our findings unravel splicing outcomes and variant-associated disruptions, enabling higher-throughput investigations into diseases and regulation mediated by RBPs.

RNA-Binding Proteins↗
PubMed2001

Simultaneous evaluation of T- and B-cell clonality, t(11;14) and t(14;18), in a single reaction by a four-color multiplex polymerase chain reaction assay and automated high-resolution fragment analysis: a method for the rapid molecular diagnosis of lymphoproliferative disorders applicable to fresh frozen and formalin-fixed, paraffin-embedded tissues, blood, and bone marrow aspirates.

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of >90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta-globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

B-Lymphocytes↗
PubMed2001

API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.

Malignant lymphoma of mucosa-associated lymphoid tissue (MALT) type is a distinct clinicopathological disease entity in the category of extranodal marginal zone B-cell lymphoma. Recently, we and others have shown that the API2 gene on chromosome 11 and the MALT1 gene on chromosome 18 are fused as a result of t(11;18)(q21;q21) in MALT lymphomas. Here we report a detection assay that can be used for formalin-fixed, paraffin-embedded specimens. It consists of a multiplex one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) followed by three parallel multiplex nested polymerase chain reactions. Eight variants of the fusion transcripts have been reported to date. When these variants were used as positive controls, all were successfully detected. The subsequent direct sequencing confirmed the results. Using this rapid and simple method, we could detect API2-MALT1 fusion transcripts in 5 of 15 (33%) archival cases of MALT lymphoma for a frequency comparable with those of RT-PCR assays using frozen materials. The lung was the preferential anatomical site of origin of MALT lymphomas harboring API2-MALT1 fusion. No fusion transcript was detected in any of 20 high-grade B-cell lymphomas. Our multiplex RT-PCR assay, which can be used for routinely-processed paraffin samples, should serve as a useful molecular tool for clarifying the clinicopathological significance of API2-MALT1 fusion in MALT lymphoma.

Adult↗
PubMed2001

Multiplex PCR assay for screening deletions in the low density lipoprotein receptor gene.

BACKGROUND: In different populations of the world, more than 150 genetic alterations of the LDL receptor gene have been identified; each of which can result in hypercholesterolaemia, but no hot spots in the gene were detected so far. Because of the existence of very variable genetic alterations in different ethnic communities, none of the assays developed for screening mutations/deletions in a population defined can be adapted to study the possible genetic defects. The present study was designed to develop a new, multiplex PCR-based, molecular biological method to screen the whole coding region of the LDL receptor gene. METHODS: Using primer pairs completely flanking the promoter and the entire exonal region, in the PCR reactions 83-386-bp long, DNA sequences were synthesised in seven different reaction mixtures. The reaction conditions of the multiplex PCR system were optimised in order to synthesise all exons and the promoter region of the gene using only two annealing temperatures. The products could be visualised separately by agarose gel electrophoresis/ethidium bromide staining. RESULTS: A rapid, effective test enabling the screening of DNA alterations in the entire LDL receptor gene was developed. Using this simple multiplex PCR assay, deletions affecting more than 10 bp in any part of the gene can be easily detected by a single agarose gel electrophoresis. CONCLUSIONS: The simplicity, specificity and versatility of the assay make it suitable system for routine screening of LDL receptor gene mutations in large population samples. This PCR assay can be recommended for screening of LDL-RG deletions in populations or groups at high risk for cardiovascular diseases.

Electrophoresis, Agar Gel↗
PubMed1999

A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella.

We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognised genotypes of Trichilnella. Partial DNA sequence data were generated from internal transcribed spacers ITS1 and ITS2, and from the expansion segment V region of the ribosomal DNA repeat from five species of Trichinella and two additional genotypes, designated T5 and T6. Five different PCR primer sets were identified which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern for each species and genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite genotype consist of no more than two well-defined DNA fragments, except isolates of T. pseudospiralis which generate multiple, closely migrating bands. The expansion segment V-derived primer set contributes at least one fragment to each genotypic pattern and, therefore, functions both as a means for differentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were verified using multiple geographical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae using a nested, multiplex PCR, whereby the entire internal transcribed spacer region as well as the gap region of the expansion segment V of the large subunit ribosomal DNA are amplified concurrently in a first-round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis.

Animals↗
PubMed2001

A new multiplex PCR strategy for the simultaneous determination of four genetic polymorphisms affecting HIV-1 disease progression.

The CCR5 Delta32, CCR2 64I, SDF1 3'A, and CCR5 promoter 59029 polymorphisms have been suggested to influence HIV-1 disease progression. Furthermore, the CCR5 Delta32 and the CCR2 64I polymorphisms have been associated with various other diseases. The purpose of the present study was to develop a multiplex assay for the simultaneous determination of these four polymorphisms. Results obtained with the multiplex assay were compared to results obtained by conventional RFLP-PCR and no differences were observed. The multiplex assay offers a quick tool for the determination of the CCR5 Delta32, CCR2 64I, SDF1 3'A, and CCR5 promoter 59029 A/G polymorphisms.

Chemokine CXCL12↗