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PubMed2006

Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA).

OBJECTIVES: To evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene. DESIGN AND METHODS: The sensitivity and accuracy of MLPA were assessed and compared with the multiplex PCR in a total of 63 subjects including 43 subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and 20 female carriers. RESULTS: MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR. In addition, the extent of the deletions and duplications could be more accurately defined which in turn facilitated a genotype-phenotype correlation. CONCLUSIONS: MLPA is superior to multiplex PCR. It should be the method of choice for the detection of exon deletions and duplications of the DMD gene in patients with DMD or BMD, as well as in female carriers.

Dystrophin↗

PubMed2005

Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes.

A single nucleotide polymorphism (SNP) multiplex has been developed to analyse highly degraded and low copy number (LCN) DNA template, i.e. <100 pg, for scenarios including mass disaster identification. The multiplex consists of 20 autosomal non-coding loci plus Amelogenin for sex determination, amplified in a single tube PCR reaction and visualised on the Applied Biosystems 3100 capillary electrophoresis (CE) system. Allele-specific primers tailed with shared universal tag sequences were designed to speed multiplex design and balance the amplification efficiencies of all loci through the use of a single reverse and two differentially labelled allele denoting forward universal primers. As the multiplex is intended for use with samples too degraded for conventional profiling, a computer program was specifically developed to aid interpretation. Critical factors taken into account by the software include empirically determined extremes of heterozygous imbalance (Hb) and the drop-out threshold (Ht) defined as the maximum peak height of a surviving heterozygous allele, where its partner may have dropped out. The discrimination power of the system is estimated at 1 in 4.5 million, using a White Caucasian population database. Comparisons using artificially degraded samples profiled with both the SNP multiplex and AMPFISTR SGM plus (Applied Biosystems) demonstrated a greater likelihood of obtaining a profile using SNPs for certain sample types. Saliva stains degraded for 147 days generated an 81% complete SNP profile whilst short tandem repeats (STRs) were only 18% complete; similarly blood degraded for 243 days produced full SNP profiles but only 9% with STRs. Reproducibility studies showed concordance between SNP profiles for different sample types, such as blood, saliva, semen and hairs, for the same individual, both within and between different DNA extracts.

Amelogenin↗

PubMed2005

Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR.

BACKGROUND: Adequate laboratory diagnosis of human rhinoviruses (hRV) and human metapneumoviruses (h MPV) requires molecular methods as viral culture lacks sensitivity. However, setting up individual PCRs for all respiratory viruses is not practical so preferentially multiplex PCRs are used. OBJECTIVES: To develop for routine diagnosis a rapid real-time PCR assay for detection of hRV and h MPV including an internal control in a single tube multiplex reaction using probes carrying different fluorophores to discriminate targets. STUDY DESIGN: The multiplex real-time RNA PCR was optimized to include the internal control virus and a total of 358 respiratory samples from 239 patients taken over a one-year period were analyzed by the multiplex assay. RESULTS: The multiplex assay with co-amplification of the internal control was as sensitive and specific as the individual assays. Application of this assay on clinical samples from 239 patients in a one-year period resulted in an incidence of hRV and h MPV of 41/239 (17.1%) and 6/239 (2.5%), respectively. Inhibition, defined as poor internal control amplification, was detected in 8 (2.2%) samples. Culture was performed on these samples and only four hRV were detected. CONCLUSIONS: This real-time PCR method enables sensitive diagnosis of these two respiratory pathogens with the potential to expand the assay as part of a full molecular respiratory viral screen.

Adolescent↗

PubMed2006

Multiplex polymerase chain reaction detection of black-pigmented bacteria in infections of endodontic origin.

The purpose of this study was to detect the presence of Porphyromonas endodontalis, P. gingivalis, Prevotella intermedia, P. nigrescens, and P. tannerae from clinical samples using multiplex polymerase chain reactions (PCR). Two different multiplex PCR protocols were used (one for the two Porphyromonas species and the other for the three Prevotella species), each one using a primer pair specific for each target species. The results were compared to those of the conventional culture procedures. Microbial samples were taken aseptically from 40 infected root canals and abscesses from patients. Samples were cultured in an anaerobic condition for conventional identification using a Rapid ID 32 A kit. Multiplex PCR was processed using the DNA extracted from each sample. At least one of the five species of black-pigmented bacteria (BPB) were detected in 65% (26 of 40) of the samples using multiplex PCR, and in 15% (6 of 40) using the conventional culture procedures. Multiplex PCR was more rapid, sensitive, specific, and effective in detecting BPB than the conventional culture procedures.

Bacteroidaceae Infections↗

PubMed2005

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses.

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

Child↗

PubMed2007

Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of human Cytomegalovirus in clinical specimens.

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.

Cytomegalovirus↗

PubMed2006

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping.

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.

Alleles↗

PubMed2004

Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. Oligonucleotide primers for the amplification of the DNA of these three organisms were optimized for use in combination in the same reaction. PCR products were detected by the Micro-Chip Electrophoresis Analysis System. Clinical samples were obtained from 208 community-acquired pneumonia (CAP) patients who were participants in a multicenter CAP surveillance study performed at seven medical schools and their affiliate hospitals in Japan. No significant differences in the sensitivity of each primer set were observed when tested in both the multiplex and monoplex PCR assays. Our multiplex PCR was able to reliably detect 10 copies/100 microl of each of the three pathogen DNAs. Of the panel of 208 samples, 14 of 15 C. pneumoniae, 10 of 10 M. pneumoniae, eight of eight L. pneumophila and 165 of 176 negative samples were correctly identified. Eleven cases who were the multiplex PCR positive and conventional method negative were observed. The PCR findings were of possible significance in at least four of these patients. Our multiplex PCR assay could potentially be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical usefulness.

Adolescent↗

PubMed2000

Use of multiplex PCR patterns as genetic markers for Burkholderia pseudomallei.

A simple PCR-based typing method was developed to differentiate between strains of Burkholderia pseudomallei. Two pairs of primers, based on sequences from two specific DNA probes, were used to amplify the bacterial DNA by multiplex PCR. We evaluated the PCR method for epidemiological typing of B. pseudomallei and compared this with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods. In RFLP, the DNA of B. pseudomallei was digested with HindIII and the pKKU-S23L was used as a probe while 5' GTTTCGCTCC 3' primer was used in RAPD. DNA was obtained from 37 B. pseudomallei environmental and clinical isolates from humans or animals. These isolates were also classified by their ability to assimilate L-arabinose. A total of 21 type patterns were identified by multiplex PCR. Among human and animal isolates, multiplex PCR revealed ten types, all of which were arabinose negative (Ara-), whereas six of the 11 types of environmental isolates were Ara-. There are two environmental patterns that also were found in clinical isolates. The RFLP technique showed 12 different types in the 37 isolates, and three of these contained both Ara+ and Ara- isolates. The RAPD technique revealed 33 different types in the 37 isolates. Multiplex PCR, therefore, is the genetic marker that best correlates with the ability of the organism to assimilate L-arabinose. Moreover, two types (M4, M15) correlated with disseminated septicemic melioidosis in the northeast Thailand. If a greater number of isolates are tested, the multiplex PCR technique may prove to be useful for rapid epidemiological typing of B. pseudomallei.

Animals↗

PubMed2001

Differentiation of porcine circovirus (PCV)-1 and PCV-2 in boar semen using a multiplex nested polymerase chain reaction.

A multiplex nested polymerase chain reaction (PCR) was developed for the detection of and differentiation between porcine circovirus (PCV)-1 and PCV-2 in boar semen. Eighteen (30%) and 30 (50%) out of 60 whole semen samples were found to be positive for PCV using multiplex conventional PCR and multiplex nested PCR, respectively. Of the 30 positive samples obtained using multiplex nested PCR, two were found to be positive for PCV-1 only, eight for PCV-2 only, and 20 for PCV-1 and PCV-2. When the separated fractions of PCV-contaminated semen were analyzed using multiplex nested PCR, PCV DNA was found to be present mainly in the seminal fluid and nonsperm cell fractions. When compared with the virus isolation method commonly used to detect viruses, this PCR assay was found to be more sensitive and rapid and, as such, may prove to be a good alternative method for the detection of and differentiation between PCV-1 and PCV-2 in boar semen.

Animals↗

PubMed2002

Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction.

We developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) to detect six citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV) and Citrus viroid OS (CVd-OS) and Apple stem grooving virus (ASGV, synonym: Citrus tatter leaf virus (CTLV)) from citrus plants. The multiplex RT-PCR was also designed to distinguish CVd-I-LSS (a distinct variant of CBLVd) from CBLVd. By the multiplex RT-PCR, one to eight fragments specific to the pathogens were simultaneously amplified from one sample and identified by their specific molecular sizes in 6% polyacrylamide gel electrophoresis. The results of the multiplex RT-PCR were consistent with those of other diagnoses, such as uniplex RT-PCR, to detect each of the pathogens. The multiplex RT-PCR provides a simple and rapid method for detecting various viroids and ASGV in citrus plants, which will help diagnose many citrus plants at a time.

Citrus↗

PubMed2003

Multiplex RT-PCR-ELISA compared with bioassay for the detection of four apple viruses.

A sensitive and reliable multiplex RT-PCR-ELISA technique for the detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus was developed. This technique is compared with the method used commonly for indexing by woody indicators, which is time consuming and expensive. For the RT-PCR-ELISA technique, the amplified products were labeled with digoxigenin during the RT-PCR by incorporation of a digoxigenin labeled primer. After hybridization of the PCR products to specific capture oligonucleotides, which were bound covalently to the surface of NucleoLink strips, anti-digoxigenin antibodies were used for detection. More than 100 samples were tested in parallel by indexing and multiplex-RT-PCR-ELISA. All infections detected by woody indicators were also detected by multiplex RT-PCR-ELISA. Furthermore, additional infections were only found by multiplex RT-PCR-ELISA. The colourimetric detection of multiplex-RT-PCR products was at least as sensitive and sometimes slightly more sensitive than detection by gel electrophoresis. The results show that this molecular technique is more reliable for the detection of the above mentioned apple viruses than indexing by woody indicators, thereby helping to reduce cost and time during the certification of plant material.

Biological Assay↗

PubMed2003

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers.

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.

Bacteriological Techniques↗

PubMed1997

Multiplex PCR for the identification of Arcobacter and differentiation of Arcobacter butzleri from other arcobacters.

A multiplex polymerase chain reaction (PCR) assay to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters is described. The test uses two primer sets. Set I targets a section of the 16S rRNA genes of Arcobacter spp. Set II amplifies a portion of the 23S rRNA genes unique to A. butzleri. Specificity of the primer sets was evaluated using ATCC reference strains of A. butzleri, A. cryaerophilus, A. skirrowii, Bacteroides spp., Campylobacter spp., Helicobacter spp. and Wolinella succinogenes. Upon PCR amplification, all of the Arcobacter isolates yielded a 1223 bp product, whereas A. butzleri ATCC 49616 exhibited both a 1223 bp and a 686 bp product. No PCR product was observed for other closely related ATCC strains (n = 37). We next analyzed by multiplex PCR field strains of Arcobacter spp. (n = 108) which had been previously characterized to the species level by either DNA-DNA hybridization, dot blot hybridization, ribotyping or by serology. The 1223 bp multiplex PCR product identified all of the isolates as Arcobacter. The presence of both the 1223 and 686 bp amplicons identified 66 strains as A. butzleri. Speciation by multiplex PCR agreed with results obtained by the other methods. The multiplex PCR assay is specific, rapid and easy to interpret and, thus, will aid in elucidating the prevalence, epidemiology and zoonotic potential of Arcobacter.

Animals↗

PubMed2001

A sixteen-channel multiplexing upgrade for single channel receivers.

With the increasing interest in phased arrays in magnetic resonance imaging, imaging system receivers capable of acquiring larger number of parallel signals are needed. Suggested techniques for rapid imaging propose the use of arrays with as many as 128 elements. While simply duplicating the number of receiver chains as needed is a viable technique, it quickly becomes both cumbersome and expensive. Time domain multiplexing offers an alternative solution to this problem. By using RF multiplexing switches, a single receiver can be upgraded to an array receiver capable of multi-channel data acquisition giving users array capability. Additionally, it can be used to dramatically increase acquisition capability of multiple receiver systems. This paper reports results from a multiplexing system upgrade, which converts a single channel standard clinical imaging system to a 16-channel array system. The upgrade includes both the RF multiplexing front-end and an external data acquisition system with image processing capability. Issues concerning the implementation of high channel-count multiplexers are also discussed.

Equipment Design↗

PubMed2003

Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions.

Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR.

Biotechnology↗

PubMed1995

Establishment and evaluation of a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex.

OBJECTIVE: To establish a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex and to evaluate the test in the diagnosis of tuberculosis. DESIGN: Three sets of primers were used to amplify 383 bp, 240 bp and 131 bp DNA fragments from the genes encoding the 65 kDa, MPB64 and the 19 kDa proteins of M. tuberculosis in a single reaction tube. Reaction conditions were optimized with respect to the requirement of DMSO, concentration of MgCl2, annealing and denaturation temperatures and number of amplification cycles. Inhibitory activity in clinical samples was identified by amplifying a 500 bp DNA fragment of the phage lambda along with the mycobacterial targets within the same reaction tube. The multiplex PCR was evaluated in differentiating M. tuberculosis complex from other mycobacteria and in the diagnosis of tuberculosis by testing clinical specimens. RESULTS: Amplification of the 383 bp DNA fragments was specific to the genus Mycobacterium. The 240 bp DNA fragment was amplified from M. tuberculosis complex and M. fortuitum and the 131 bp DNA fragment was amplified from the mycobacteria of M. tuberculosis complex and M. scrofulaceum. All the three bands were amplified only from M. tuberculosis complex. Applicability of the multiplex PCR is demonstrated in differentiating M. tuberculosis complex from other mycobacteria by using standard strains and clinical isolates. The multiplex PCR was also useful in the detection of inhibitory activity and in the identification of M. tuberculosis complex directly in clinical samples. CONCLUSION: The multiplex PCR established in this study could differentiate M. tuberculosis complex from other mycobacteria. This test may also be helpful in the early and specific diagnosis of tuberculosis.

Bacterial Typing Techniques↗

PubMed2004

NIST mixed stain study 3: signal intensity balance in commercial short tandem repeat multiplexes.

Short-tandem repeat (STR) allelic intensities were collected from more than 60 forensic laboratories for a suite of seven samples as part of the National Institute of Standards and Technology-coordinated 2001 Mixed Stain Study 3 (MSS3). These interlaboratory challenge data illuminate the relative importance of intrinsic and user-determined factors affecting the locus-to-locus balance of signal intensities for currently used STR multiplexes. To varying degrees, seven of the eight commercially produced multiplexes used by MSS3 participants displayed very similar patterns of intensity differences among the different loci probed by the multiplexes for all samples, in the hands of multiple analysts, with a variety of supplies and instruments. These systematic differences reflect intrinsic properties of the individual multiplexes, not user-controllable measurement practices. To the extent that quality systems specify minimum and maximum absolute intensities for data acceptability and data interpretation schema require among-locus balance, these intrinsic intensity differences may decrease the utility of multiplex results and surely increase the cost of analysis.

Alleles↗