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PubMed2002

Rapid and specific identification of Shiga toxin-producing Escherichia coli in faeces by multiplex PCR.

AIMS: The object of this study was to develop a multiplex PCR system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) in faeces. METHODS AND RESULTS: A multiplex PCR (mPCR) protocol was developed using a primer pair specific for genes that are involved in the biosynthesis of the O157 E. coli antigen, and primers that identify the sequences of Shiga toxin 1 and 2 (stx 1 and stx1) and the intimin protein (eaeA). The mPCR assay was used for amplification of STEC genes in bacteria directly (after enrichment) in faeces. The test was very sensitive and could detect between 9 and 1 bacterial cells per gram of faeces. The mPCR was used for the examination of 69 bovine faecal samples derived from healthy cattle. The results indicated that 62 x 3% of the samples were positive, generating at least one PCR amplicon of the expected size. CONCLUSIONS: The method can be applied for rapid and specific identification of STEC bacteria in faecal samples, and for differentiation of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect Shiga toxin-producing E. coli directly in faeces within a short time represents a considerable advancement over more time-consuming and less sensitive methods for identification and characterization of STEC bacteria.

Animals↗
PubMed2002

Determination of Helicobacter pylori vacA allelic types by single-step multiplex PCR.

AIMS: To develop and evaluate a novel multiplex PCR assay that enables definition of Helicobacter pylori vacA allelic type in a single reaction. METHODS AND RESULTS: Application of the one-step system to DNA extracts from 22 cultures of known vacA genotype demonstrated that it was highly accurate. Analysis of 15 matched gastric biopsy/culture pairs generated exactly correlating genotype profiles. vacA genotypes were determined from an additional 62/70 gastric biopsies from dyspeptic patients of known H. pylori positive status by the one-step assay, compared with 63/70 by the original two-reaction test. Types s1/m1, s1/m2 and s2/m2 were identified in 51.9%, 31.2% and 16.9% of biopsies, respectively. CONCLUSIONS: The multiplex PCR system developed enables rapid one-step vacA genotyping that is accurate, easy to interpret and more economical than the alternative multiple-reaction tests. Application of this system to gastric biopsies from patients in South-east England demonstrated that s1/m1 was the most common genotype, while s1/m2 and s2/m2 were less prevalent. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple one-step system can be applied direct to antral gastric biopsies without the need for culture, thereby facilitating rapid surveillance of vacA genotype in relation to geographical location and disease status.

Alleles↗
PubMed1996

Genotyping of the human platelet antigen systems 1 through 5 by multiplex polymerase chain reaction and ligation-based typing.

BACKGROUND: Platelet-specific antibodies may be involved in refractoriness to platelet transfusions, disorders such as neonatal alloimmune thrombocytopenia, and post-transfusion purpura. Genotyping for the major human platelet antigen (HPA) systems HPA-1 through HPA-5 is of considerable help in establishing the diagnoses of these diseases. STUDY DESIGN AND METHODS: A new genotyping method is described. Alleles of all five systems are amplified in a multiplex polymerase chain reaction. Subsequently, aliquots of the amplification products are thermocycled in the presence of a pair of allele-specific oligonucleotide probes and a heat-stable ligase. After heat denaturation, the probes hybridize adjacent to complementary sequences of the amplification product. In a perfect match, the two probes become covalently joined. Detection of the ligation product is performed with an enzyme-linked immunosorbent assay. RESULTS: Complete concordance of genotypes between the ligation-based typing and established genotyping methods was determined in 54 Austrian (HPA-1, -2, -3, and -5) and 56 Japanese (HPA-4) individuals. Ligation-based genotyping of HPA-1 polymorphism using platelet-derived RNA as starting material gave concordant results in all 15 cases tested. CONCLUSION: Multiplex polymerase chain reaction in combination with ligation-based typing allows fast typing of large numbers of platelet donors and screening for critical antigens in pregnant women.

Base Sequence↗
PubMed2003

Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions.

Detection of causal mutations is required for genetic counseling. Molecular modeling combined with patients' phenotype provides significant insight into structure-function relationship of factor (F)VIII molecule. Our objective was to identify defects in the gene of FVIII by a sensitive and simple scanning technique with high throughput in order to study molecular mechanisms by which novel amino acid substitutions may lead to hemophilia A. A cohort of 81 families with mild, moderate and severe hemophilia A negative in intron 22 inversion was studied. For detection of mutations in the FVIII gene a conformation sensitive gel electrophoresis (CSGE) was modified by multiplexing. Thirteen novel amino acid substitutions were studied by molecular modeling and a correlation with the cross-reactive material (CRM) phenotype was performed. In 74 families, 59 different mutations were detected. Six different mutations were recurrent in 21 unrelated families. Thirty-four novel mutations included 19 point mutations, four small insertions, nine small deletions and two complex mutations. Thirteen novel amino acid substitutions occurred at residues conserved in FVIII orthologs. Five of them were associated with a discrepancy between FVIII activity and antigen; another five with CRM reduced phenotype and one with undetectable FVIII antigen. Multiplexing of the CSGE significantly increased its throughput without substantial loss of sensitivity. Molecular modeling suggested mechanisms by which substitutions at residues 382 and 569, located outside the proposed FIXa-binding region, may influence FVIII/FIXa interaction. His2155 was predicted to participate in FVIII/VFW binding.

Amino Acid Substitution↗
PubMed2003

Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci.

In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing result that enhances the reliability of the consensus profile, which is commonly retrieved from two separate 34 cycle PCR results. Furthermore, the 28 + 6 cycles approach may be used to screen LCN samples for their potential to produce a 34 PCR cycle profile. Finally and as a last resort the 28 + 6 cycles approach can be used in those cases where no further extract from the crime sample is available. Finally, the potential of LCN typing was demonstrated in typing samples from non-probative and actual casework examples. From a high proportion of samples that failed to demonstrate SGM plus typing results using the standard protocol of 28 cycles, at least partial profiles could be obtained after LCN methods were used. For example, LCN typing was applied in a case where 10-year old samples from bones and teeth that were retrieved from a mass grave had to be identified. This study resulted in the positive identification of a number of victims by comparing the LCN DNA profiles with the profiles from putative relatives. The value of LCN DNA typing was further demonstrated in a strangulation case. The throat of the victim was sampled and only after 34 PCR cycles were we able to reveal that the evidential sample contained a distinct mixture of the victim's own DNA and the DNA of the defendant.

Consensus Sequence↗
PubMed2000

Caesarean section using a combined spinal epidural technique in a patient with arthrogryposis multiplex congenita.

A case of a woman with arthrogryposis multiplex congenita presenting for elective caesarean section is reported. A combined spinal epidural anaesthetic technique was used. Aetiology and anaesthetic considerations for patients with arthrogryposis multiplex congenita are discussed. The importance of early referral to the anaesthetic team of patients with intercurrent disease or congenital syndromes is emphasised.

Journal Article↗
PubMed2000

Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes.

BACKGROUND: Prader-Willi (PWS) and Angelman's (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively simple alternative method to detect the methylation status of the PWS/AS region. METHODS AND RESULTS: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. CONCLUSION: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.

Angelman Syndrome↗
PubMed1996

Development and Validation of a Multiplex PCR-Based Assay for the Upper Respiratory Tract Bacterial Pathogens Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.

Background: Conventional simplex polymerase chain reaction (PCR)-based assays are limited in that they only provide for the detection of a single infectious agent. Many clinical diseases, however, present in a nonspecific, or syndromic, fashion, thereby necessitating the simultaneous assessment of multiple pathogens. Panel-based molecular diagnostic testing can be accomplished by the development of multiplex PCR-based assays, which can detect, individually or severally, different pathogens that are associated with syndromic illness. As part of a larger program of panel development, an assay that can simultaneously detect Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was developed. These organisms were chosen as they are the most common bacterial pathogens associated with both the acute and chronic forms of otitis media; they are also responsible for a high percentage of sinus infections in both children and adults. In addition, H. influenzae and S. pneumoniae are commonly associated with septic meningitits. Methods and Results: Multiple individual PCR-based assays were developed for each of the three target organisms which were then evaluated for sensitivity and specificity. Utilizing the simplex assays that met our designated performance criteria, a matrix style approach was used to develop a duplex H. influenzae-S. pneumoniae assay. The duplex assay was then used as a single component in the development of a triplex assay, wherein the various M. catarrhalis primer-probe sets were tested for compatibility with the existing assay. A single-step PCR protocol, with species-specific primers for each of the three target organisms and a liquid hybridization-gel retardation amplimer detection system, was developed, which amplifies and then discriminates among each of the amplification products according to size. This assay is able to detect all three organisms in a specific manner, either individually or severally. Dilutional experiments indicate a detection limit of 10 femtograms (fg)(6-7 genomic equivalents) or less of genomic DNA for each of the three microorganisms regardless of the presence of irrelevant DNA. Conclusions: The reliance on individual, robust, species-specific primers and the avoidance of a nested PCR approach make this bacterial multiplex assay suitable for use in the clinical laboratory. This assay has proved useful in both research and patient care applications.

Journal Article↗
PubMed1988

Arthrogryposis multiplex congenita associated with lissencephaly: a case report.

A child with arthrogryposis multiplex congenita and microcephaly is described. Cranial CT-scan and MRI showed abnormalities consistent with type I lissencephaly. The lissencephaly seems to be the primary cause of the congential contractures. Lissencephaly associated with arthrogryposis multiplex congenita has to be considered as a special kind of lissencephaly syndrome.

Arthrogryposis↗
PubMed1985

EMG and needle muscle biopsy studies in arthrogryposis multiplex congenita.

From 1979 to 1983, EMG and/or muscle biopsy studies were done in twenty-one of twenty-two patients seen in our institution for the first time for arthrogryposis multiplex congenita (AMC). Most of the muscle biopsies were done with a needle. As expected, the final diagnosis in these patients was very heterogeneous since it is well recognized that AMC can be due to any factor that interferes with fetal mobility. We found however a surprisingly high number of patients (9/22) affected by a myopathic disease. Based on these results, we feel that needle muscle biopsy should be included as a routine procedure in infants affected by AMC since by clarifying the diagnosis it helps in improving genetic counselling and prognosis in these patients. It is now widely accepted that arthrogryposis multiplex congenita (AMC) is not in itself a disease but is secondary to any factor that interferes with fetal movements. The joint deformities seen in these children can be due to mechanical factors (oligohydramnios for example), central or peripheral nervous system disorders, muscle diseases... (Dubowitz 1978, Hageman and Willemse 1983, Hall 1981, Swinyard 1982). However, the relative frequency of each etiologic factor remains unclear since in spite of the abundant literature on the subject, we found only three reports describing the findings of electromyographic (EMG) and muscle biopsy studies done systematically in unselected groups of children affected by AMC (Amick et al 1967, Bharucha et al 1972, Dastur et al 1972, Kullmann and Szijj 1973). Furthermore, each article reached different conclusions in regard to the occurrence of these various etiologies in their respective patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthrogryposis↗
PubMed1990

Arthrogryposis multiplex congenita; feeding, language and other health problems.

A survey of the health problems of 87 individuals with arthrogryposis multiplex congenita revealed a high proportion (51) with major feeding difficulties in infancy. This was generally related to structural abnormalities of the jaw and tongue. Children thus involved were more prone to recurrent chest infections, constipation, and poor growth and weight gain. In addition, there was a strong association between initial feeding difficulties and subsequent language problems. Early identification of a sub-group of children with arthrogryposis multiplex congenita is therefore possible who may require continuing therapy from a number of disciplines.

Arthrogryposis↗
PubMed2005

Chemical genomic profiling to identify intracellular targets of a multiplex kinase inhibitor.

The identification of the kinase or kinases targeted by protein kinase inhibitors is a critical challenge in validating their use as therapeutic agents or molecular probes. Here, to address this problem, we describe a chemical genomics strategy that uses a direct comparison between microarray transcriptional signatures elicited by an inhibitor of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered candidate kinase targets. By using this approach, we have identified two cyclin-dependent kinases, Cdk1 and Pho85, as the targets of the inhibitor GW400426 in Saccharomyces cerevisiae. We demonstrate that simultaneous inhibition of Cdk1 and Pho85, and not inhibition of either kinase alone, by GW400426 controls the expression of specific transcripts involved in polarized cell growth, thus revealing a cellular process that is uniquely sensitive to the multiplex inhibition of these two kinases. Our results suggest that the cellular responses induced by multiplex protein kinase inhibitors may be an emergent property that cannot be understood fully by considering only the sum of individual inhibitor-kinase interactions.

CDC2 Protein Kinase↗
PubMed2003

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens.

PURPOSE: A majority of ocular viral diseases are caused by herpes group of viruses. Such infections, especially atypical herpetic keratitis, iridocyclitis and intra-ocular inflammations, can often present with overlapping clinical manifestations misleading the diagnosis. Molecular techniques are most useful in such instances for an accurate and rapid diagnosis since conventional methods are time consuming and less sensitive. A multiplex PCR was developed and used for the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) in ocular samples. METHODS: One hundred and forty six ocular samples (corneal scrapings - 52, aqueous fluid - 36, vitreous fluid - 31, tissues - 26, skin vesicle scraping - 1) were included in the study. The sensitivity of the assay was determined using serial dilutions of standard strains of HSV, VZV, and CMV vis-à-vis plaque forming assay. RESULTS: The sensitivity of the assay was 4, 4 and 12 PFU/ml or 20, 20 and 60 genome copy numbers of HSV, VZV and CMV respectively. Using DNA from various sources (fungal, bacterial, human leukocytes, tissues) along with standard positive controls, the assay was found to be highly specific. HSV DNA was detected in majority of the clinical samples (33.6%), most frequent being corneal samples. Comparatively, VZV and CMV infections were detected in small number of samples (VZV-3, CMV-2). CONCLUSIONS: We found the assay very useful in our set-up whenever a differential diagnosis of herpetic infections was suggested by the ophthalmologist. The multiplex PCR we have described here can be of greater value in clinics with larger number of patients suspected of having HSV, VZV or CMV infections.

Cytomegalovirus↗
PubMed2004

Xylella fastidiosa subspecies: X. fastidiosa subsp. [correction] fastidiosa [correction] subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov.

Xylella fastidiosa, a fastidious bacterium causing disease in over 100 plant species, is classified as a single species, although genetic studies support multiple taxons. To determine the taxonomic relatedness among strains of X. fastidiosa, we conducted DNA-DNA relatedness assays and sequenced the 16S-23S intergenic spacer (ITS) region using 26 strains from 10 hosts. Under stringent conditions (Tm -15 degrees C), the DNA relatedness for most X. fastidiosa strains was *70%. However, at high stringency (Tm -8 degrees C), three distinct genotypes (A, B, and C) were revealed. Taxon A included strains from cultivated grape, alfalfa, almond (two), and maple, interrelated by 85% (mean); taxon B included strains from peach, elm, plum, pigeon grape, sycamore, and almond (one), interrelated by 84%; and taxon C included only strains from citrus, interrelated by 87%. The mean reciprocal relatedness between taxons A and B, A and C, and B and C, were 58, 41, and 45%, respectively. ITS results also indicated the same grouping; taxons A and B, A and C, and B and C had identities of 98.7, 97.9, and 99.2%, respectively. Previous and present phenotypic data supports the molecular data. Taxon A strains grow faster on Pierce's disease agar medium whereas B and C strains grow more slowly. Taxon B and C strains are susceptible to penicillin and resistant to carbenicillin whereas A strains are opposite. Each taxon can be differentiated serologically as well as by structural proteins. We propose taxons A, B, and C be named X. fastidiosa subsp. fastidiosa [correction] subsp. nov, subsp. multiplex, subsp. nov., and subsp. pauca, subsp. nov., respectively. The type strains of the subspecies are subsp. fastidiosa [correction] ICPB 50025 (= ATTC 35879T and ICMP 15197), subsp. multiplex ICPB 50039 (= ATTC 35871 and ICMP 15199), and subsp. pauca ICPB 50031 (= ICMP 15198).

Anti-Bacterial Agents↗
PubMed2000

A multiplex-PCR method to detect enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli in artificially contaminated foods.

In view of the considerable public health risk of the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, a multiplex-PCR based method was used for the amplification of the slt genes and of the eaeA gene. Three pairs of primers, different from the oligonucleotides previously used by other authors, were exploited for the amplification. Different E. coli serotypes were tested with the optimized protocol. Fifty three artificially contaminated samples and sixty naturally contaminated samples were processed with the multiplex-PCR. All the artificially contaminated samples gave positive results independently of the number of cells inoculated. On the contrary, the naturally contaminated samples were all negative. The results obtained from this experiment demonstrated that this protocol could be used for monitoring the spread of these organisms in food.

DNA Primers↗
PubMed2004

A rapid RT-PCR screening assay incorporating multiplexed validated control genes for CBF rearrangements at diagnosis in AML.

AIMS: Our objective was to establish a multiplexed assay using the Biomed 1 primers to detect AML1-ETO transcripts and 10 different CBFB-MYH11 transcripts, using BCR and ABL transcripts as controls. METHODS: Control genes were systematically tested for characteristics of optimal controls. The final assay was validated on 50 AML patient samples. RESULTS: Testing confirmed that the designated control gene criteria were fulfilled. Of 50 patient samples tested, four RT-PCR results were discordant with the cytogenetic result. In three cytogenetically negative cases, RT-PCR detected cryptic CBF rearrangements (one AML1-ETO and two CBFB-MYH11). The fourth case was inv(16) positive but negative by RT-PCR; however, the control gene result revealed suboptimal RNA quality. CONCLUSIONS: We have described a robust multiplex RT-PCR assay that incorporates experimentally validated control genes that are important for accurate interpretation. The assay is more sensitive than cytogenetics in the detection of CBF AML. Application to large patient cohorts will determine the prognostic significance of cryptic CBF rearrangements compared with their cytogenetic counterparts.

Acute Disease↗
PubMed2000

Molecular epidemiology survey of toxinogenic Clostridium perfringens strain types by multiplex PCR.

Toxin genotypes of 95 C. perfringens strains collected within a 45-year period were analysed by a multiplex PCR. A set of primers designed for 4 different genes encoding the alpha, beta, epsilon, and iota toxins was used in a single reaction with a sensitivity of gene detection of 200 fg for DNA extracted from pure culture. Most of the strains (97%) conformed to the A biotype, and the remaining to the C or E biotypes. For biotype determination, seroneutralization of lethality in mice was performed by intravenous injection. Toxin phenotype and genotype profile were concordant in 94% of strains. Our results documented the presence of rare toxin genotypes of C. perfringens in a Polish geographical region and indicated the suitability of multiplex PCR as a method supplementing classical techniques and providing better insight into the prevalence of toxinogenic C. perfringens strains.

Animals↗
PubMed2005

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases.

BACKGROUND: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay. METHODS: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested. RESULTS: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92-97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively. CONCLUSION: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.

Antibodies, Antinuclear↗