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PubMed2000

Report of the European Network of Forensic Science Institutes (ENSFI): formulation and testing of principles to evaluate STR multiplexes.

This paper describes a collaborative exercise organised under the auspices of the European Network of Forensic Science Institutes (ENFSI). The purpose of this EU (European Union) funded group is to carry out research to enable STR loci to be compared between European laboratories, ultimately leading to the formation of a pan-European database. Accordingly, an exercise was designed to evaluate a prototype STR multiplex system manufactured by Applied Biosystems (ABD). Each laboratory was sent 12 samples to analyse along with a multiplex kit. Of specific interest was the definition of parameters to define the efficiency of the system. Stutter, split allelic peaks (differing by one base), pull-up, heterozygous balance and between locus balance were all objectively measured. Once the important parameters are defined it is possible to directly compare performances of different multiplexes and the different laboratories carrying out the tests. Since the multiplex used was a prototype system, this exercise cannot be regarded as a proficiency test.

Alleles↗
PubMed1997

Rapid detection of different RNA respiratory virus species by multiplex RT-PCR: application to clinical specimens.

BACKGROUND: The polymerase chain reaction (PCR) applied in diagnostic and epidemiologic investigations is very useful for sensitivity, specificity and time saving. OBJECTIVE: We have developed a method for the detection of genomic RNA of two different species of virus, the influenza A virus (IA) and the respiratory syncytial virus (RS), which are responsible for clinical similarities. We applied this multiplex RT-PCR protocol on clinical specimens. STUDY DESIGN: We describe a method which allows rapid diagnosis by performing a single retro-transcriptase (RT) reaction associated with the PCR (multiplex RT-PCR) on different genomes in a single sample. We have evaluated the sensitivity and the specificity of the multiplex test on positive controls, then, on RNA extracted from clinical specimens harvested from 15 children with respiratory symptoms during the spring-winter season 1997. RESULTS AND CONCLUSIONS: The multiplex RT-PCR protocol, applied to respiratory specimens, allows the investigation of RNA IA virus and RS virus in a single sample at the same time. The detection of the etiologic viral agent is rapid and it is possible to evaluate incidental simultaneous infections.

Animals↗
PubMed2000

Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction.

We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format.

Alleles↗
PubMed2004

Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR.

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The results indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.

Base Sequence↗
PubMed2002

A flow injection on-line multiplexed sorption preconcentration procedure coupled with flame atomic absorption spectrometry for determination of trace lead in water, tea, and herb medicines.

One of the limitations in previous flow injection (FI) sorption preconcentration procedures in a knotted reactor (KR), which have been carried out exclusively with a single continuous sample injection over a certain period, is the relatively low retention efficiency (typically 40-50%). Although the sensitivity of such systems could be improved by properly increasing sample preconcentration time, sample loading flow rate, or both, further improvement of the sensitivity has been limited by the narrow linearity of the relationship between signal intensity and preconcentration time or sample loading time. In this work, a novel on-line FI multiplexed sorption preconcentration procedure with repetitive sample injections was developed to overcome the above problems in the previous systems. In contrast to previous FI preconcentration systems, the proposed multiplexed preconcentration procedure evenly divides a single longer sample injection step into several shorter substeps while the total preconcentration time is still kept constant. To demonstrate its merits, the proposed FI on-line KR multiplexed sorption preconcentration system was combined with flame atomic absorption spectrometry (FAAS) for determination of trace lead in water, tea, and herb medicines. The lead in the sample solution on-line reacted with ammonium pyrrolidine dithiocarbamate, and the resultant analyte complex was sorbed on the inner walls of the KR. The residual sample solution was then removed from the KR with an air flow. The above two steps were repeated eight times with a total preconcentration time of 120 s. The sorbed analyte was eluted from the KR with 4.5 mol L(-1) HCl for on-line FAAS detection. The present multiplexed preconcentration procedure with eight repetitive sample injections for a total preconcentration time of 120 s gave a retention efficiency of 92%, twice that obtained by one single sample injection preconcentration (47%). In addition, the linear ranges of the diagrams of absorbance against sample loading flow rate and sample loading time were extended, offering more potential for achieving high sensitivity by increasing sample loading rates or sample loading time compared to the previous one single continuous sample injection preconcentration procedure. At a sample loading flow rate of 3.6 mL min(-1) for a total preconcentration period of 120 s, an enhancement factor of 57 and a detection limit (3sigma) of 8 microg L(-1) were obtained. The precision was 1.4% (RSD, n = 11) at the 200 microg L(-1) level. The developed method was successfully applied to the determination of trace lead in various water samples, herb medicines, and a certified tea reference material.

Flow Injection Analysis↗
PubMed2002

Multiplexed, high-throughput genotyping by single-base extension and end-labeled free-solution electrophoresis.

Technologies that allow for high-throughput, economical, and accurate single nucleotide polymorphism (SNP) genotyping are becoming crucial for modern genomic efforts. Here, we present a method for multiplexed single-base extension (SBE) genotyping that takes advantage of the unique separation modalities made possible via end-labeled free-solution electrophoresis (ELFSE). Three unique SBE oligonucleotide primers, which probe for mutations of clinical importance in the human p53 gene, were covalently conjugated to three unique polypeptoid frictional end labels and mixed together. This primer-polypeptoid conjugate cocktail was then used in a multiplexed SBE reaction followed by free-solution separation in a 96-capillary array electrophoresis (CAE) instrument. The study was designed to demonstrate multiplexed SNP genotyping of several loci in a single reaction and a single subsequent analysis. Further, the electrophoretic analysis was conducted without any viscous polymeric separation media, was complete in less than 10 min, and can be implemented in any capillary or microfluidic electrophoretic system with four-color fluorescent detection capabilities. Multiplexed SBE-ELFSE genotyping analysis resulted in the simultaneous and accurate genotyping of three p53 loci on five different DNA templates in a single reaction set and single CAE analysis. With the implementation of this method in 96 or more capillaries in parallel, high-throughput screening of SNPs will be accessible to a large number of laboratories.

DNA Primers↗
PubMed2006

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection.

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.

Biological Warfare↗
PubMed2003

DNA sequence context and multiplex hybridization reactions: melting studies of heteromorphic duplex DNA complexes.

Heteromorphic hybrid duplex DNA complexes are duplex states, other than perfectly matched duplexes, that can form when single strands comprising several different perfectly matched duplexes are simultaneously present in solution. Such cross-hybridization "side reactions" are of particular nuisance in multiplex reaction schemes, where many strands are designed to hybridize in parallel fashion with only their corresponding perfect complement strand. Relative to the perfect match duplexes, the sequence dependent features of these heteromorphic duplex states and their thermodynamic stability are an important consideration for multiplex hybridization reaction design. We have measured absorbance versus temperature melting curves and performed differential scanning calorimetry measurements on various mixtures of eight different 24 base single strands. When perfect complementary pairs of strands are mixed in single reactions, four perfectly matched duplexes form. When mixtures of strands that are not perfectly matched are prepared and analyzed, melting transitions for cross-hybridization are observed along with significant hyperchromicity changes. This is indicative of a melting hybrid, heteromorphic duplex states formed from two nonperfectly matched strands. In addition, when both the perfectly matched and noncomplementary strands are mixed together (in multiplex hybridization reactions) at molar ratios of 1:1, 3:1, and 1:3, evidence of perfect duplex and heteromorphic duplex complexes is found in all cases. A new analytical tool for considering heterogeneous, duplex complexes in multiplex hybridization mixtures is presented and employed to interpret the acquired melting data.

Base Pair Mismatch↗
PubMed2004

Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence.

The simultaneous detection of Staphylococcus aureus, Listeria monocytogenes, and Salmonella spp. has been approached by a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF). As compared to slab gel electrophoresis, the use of CGE-LIF improved from 10- to 1000-fold the sensitivity of the multiplex PCR analysis, allowing the detection of 2.6 x 10(3) cfu mL(-1) of S. aureus, 570 cfu mL(-1) of L. monocytogenes, and 790 cfu mL(-1) of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu mL(-1) of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The CGE-LIF method is shown to be reproducible, providing relative standard deviation (RSD) values lower than 0.8% for analysis time and lower than 5.8% for peak areas. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way.

Electrophoresis, Capillary↗
PubMed1997

Multiplex PCR for simultaneous detection of the most frequent T cell receptor-delta gene rearrangements in childhood ALL.

A rapid and simple multiplex polymerase chain reaction (PCR) is described that is capable of identifying the six most frequent rearrangements of the T cell receptor (TCR)-delta gene segments in childhood acute lymphoblastic leukemia (ALL). The PCR products amplified in a single reaction are of different size for each TCR-delta gene rearrangement. Therefore, they are readily and unambiguously distinguished after agarose gel electrophoresis and assigned to a specific V-D-J gene rearrangement. There is no need for labor-intensive and time-consuming Southern blot hybridization or nested PCR. To evaluate the multiplex assay we chose 45 DNA samples of childhood ALL analyzed beforehand for TCR-delta gene rearrangements by Southern blot and single PCR of which 30 showed TCR-delta gene rearrangements. The multiplex PCR results corresponded to the Southern blot and single PCR analyses. The described multiplex PCR enables the detection of clonal markers in about 50% of patients in order to monitor minimal residual disease (MRD) in prospective studies with a high turnover of samples.

Blotting, Southern↗
PubMed1999

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors.

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

Colorectal Neoplasms↗
PubMed1998

Multiplex PCR for TCR delta rearrangements: a rapid and specific approach for the detection and identification of immature and mature rearrangements in ALL.

The preferential occurrence of immature T-cell receptor (TCR) delta rearrangements (i.e. incomplete Ddelta2-Ddelta3 and Vdelta2-Ddelta3) in B-cell precursor acute lymphoblastic leukaemia (BCP ALL) and of predominantly mature rearrangements (incomplete Ddelta2-Jdelta1, complete Vdelta1, Vdelta2, Vdelta3 to Jdelta1) in T-lineage ALL prompted us to establish two separate multiplex PCR systems for the identification of clonal TCRdelta rearrangements. PCR products of the expected size for the specific rearrangements were detectable from a dilution of 100-1000 clonal cells in 150000 polyclonal cells. Both multiplex PCR systems were used to analyse samples from 86 childhood BCPALLs and 30 T-lineage ALLs. The results of the multiplex PCRs were controlled by standard PCR analyses for the individual rearrangements and Southern blots, which were identical. Only immature TCRdelta rearrangements were detected in BCP ALL (59%), whereas no rearrangement was found in the remaining BCP leukaemias, thus confirming the exclusive presence of immature TCRdelta rearrangements in B-lineage cells. 50% of the T-lineage ALLs contained mature rearrangements, but no immature rearrangements were found. These two multiplex PCR techniques appear to be reliable and fast aids in the analysis of clonal TCRdelta rearrangements in ALL.

Blotting, Southern↗
PubMed2003

Development of a new sensitive and efficient multiplex polymerase chain reaction (PCR) for identification and differentiation of different mycobacterial species.

For early detection and species differentiation of mycobacteria, polymerase chain reaction (PCR) techniques are currently in wide use. However, individual techniques using amplification of different targets with appropriate primers still have some limitations, which have to be overcome. The ideal technique would use DNA sequences which should be present in all mycobacteria and absent in others and would be able to discriminate one species from the other, as non-tuberculous mycobacteria (NTM) are on rise in terms of frequency of detection. We developed a multiplex PCR based on amplification of 165, 365 and 541 bp target fragments of unrelated genes, hsp 65 coding for 65 kDa antigen, dnaJ gene of mycobacteria and insertion element IS 6110 of Mycobacterium tuberculosis, respectively. This multiplex PCR was tested over 5 years from 1996 to 2001 with 411 clinical specimens from suspected cases of tuberculosis and mycobacterioses and compared with standard laboratory techniques. The multiplex PCR was positive for 379 cases compared with 280 cases by standard techniques (P < 0.0001). It could distinguish between strains of the M. tuberculosis complex and NTM; the results are comparable with standard techniques. Thus the multiplex PCR can be useful in early detection, species differentiation and epidemiology.

Bacterial Typing Techniques↗
PubMed2003

CO2 laser therapy in a case of steatocystoma multiplex with prominent nodules on the face and neck.

BACKGROUND: Steatocystoma multiplex is an uncommon disorder which usually begins in adolescence or early adult life. The condition can be hereditary, as an autosomal dominant trait, or nonhereditary, as in this case. METHODS: A 40-year-old woman presented with a history of asymptomatic nodules that began around puberty on the face. There was no family history of similar lesions. Clinical examination revealed multiple nodules distributed on the face and neck. The histopathologic examination of a biopsy specimen showed the typical features of steatocystoma multiplex. CO2 laser therapy without anesthesia was employed, and the contents were evacuated by squeezing the cysts with a pair of forceps. RESULTS: Very good results were obtained with rapid healing, minimal invasiveness, and without anesthesia. There was no evidence of scar formation and no signs of recurrence at 2-year follow-up. CONCLUSIONS: Different treatments have been reported for steatocystoma multiplex. We consider CO2 laser therapy to be an ideal technique for the treatment of steatocystoma multiplex, especially when the lesions are localized in aesthetically important areas.

Adult↗
PubMed2001

Development of multiplexed nucleic acid testing for human immunodeficiency virus type 1 and hepatitis C virus.

BACKGROUND AND OBJECTIVES: In most Western countries, blood donations are routinely screened for hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR) or other nucleic acid tests. We describe the development of a multiplexed assay for human immunodeficiency virus type 1 (HIV-1) and HCV in an internally controlled PCR suitable for large-scale blood donor screening. MATERIALS AND METHODS: The HIV/HCV multiplexed PCR used primers from highly conserved regions in the long terminal repeat region. The National Institute for Biological Standards and Controls (NIBSC) International HIV-1 RNA standard, run control and HIV-1 subtype panel were used for assay evaluation. RESULTS: The HIV-1 PCR showed a sensitivity of 24 IU/ml for HIV-1 RNA (a dilution where 95% of replicate reactions were positive), which was at least five times more sensitive than the Roche Monitor version 1.5 (using the ultrasensitive extraction protocol) and Organon NASBA assays. The assay was capable of detecting all subtypes of HIV-1 (A to H), as well as the more divergent group N and O variants. The sensitivity of the PCR was unaffected by multiplexing with HCV primers and by the presence of a bovine viral diarrhoea virus (BVDV) internal control. CONCLUSION: We have developed a highly sensitive multiplexed PCR for HIV-1 and HCV RNA screening that can be introduced into current PCR-based blood donor screening at minimal cost and without significant operational changes.

Conserved Sequence↗
PubMed2001

Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD.

AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD). METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR. Two distinct ERIC-PCR and RAPD fragments, which separated B. anthracis into two groups, were used as probes in Southern hybridization experiments. The probes hybridized only to the cya+ B. anthracis strains identified by the multiplex PCR. Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B. anthracis. CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B. anthracis virulence factors. ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B. anthracis strains and separated them from the closely related B. cereus group bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B. anthracis. Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay.

Bacillus anthracis↗
PubMed2003

Retrospective study of a plague outbreak by multiplex-PCR.

AIMS: To determine the effectiveness of multiplex-PCR in Yersinia pestis identification in samples preserved in Cary & Blair medium and to evaluate if this technique would uncover Y. pestis-positives among culture-negative samples. METHODS AND RESULTS: Multiplex-PCR was used to detect Y. pestis in Cary & Blair preserved bubo aspirates from experimentally infected guinea pigs and to re-analyze samples from a plague outbreak after prolonged storage in Cary & Blair. Variation in the target genes amplification was observed over time. CONCLUSIONS: Multiplex-PCR proved to be more effective than culture for plague diagnosis, both for old and recent samples. This technique would be a valuable tool for the plague control programme. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex-PCR technique can be useful for the detection and characterization of Y. pestis even when the bacteria are no longer viable and when culture diagnosis has been hampered by the growth of contaminants.

Animals↗
PubMedDate not supplied

Congenital linear steatocystoma multiplex of the nose.

We describe a case of steatocystoma multiplex with an unusual clinical manifestation. A 5-year-old girl had 8 papulonodular lesions arranged in a linear fashion on the left side of the nose. These had been present at birth. Histologically the lesions showed the typical features of steatocystoma multiplex. There was no family history of similar lesions. This is the second case of linear steatocystoma multiplex, a rare variant of steatocystoma multiplex.

Child, Preschool↗