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PubMed2004

Minimal 16q genomic loss implicates cadherin-11 in retinoblastoma.

Retinoblastoma is initiated by loss of both RB1 alleles. Previous studies have shown that retinoblastoma tumors also show further genomic gains and losses. We now define a 2.62 Mbp minimal region of genomic loss of chromosome 16q22, which is likely to contain tumor suppressor gene(s), in 76 retinoblastoma tumors, using loss of heterozygosity (30 of 76 tumors) and quantitative multiplex PCR (71 of 76 tumors). The sequence-tagged site WI-5835 within intron 2 of the cadherin-11 (CDH11) gene showed the highest frequency of loss (54%, 22 of 41 samples tested). A second hotspot for loss (39%, 9 of 23 samples tested) was detected within intron 2 of the cadherin-13 (CDH13) gene. Furthermore, deletion of the exons of CDH11 and/or WI-5835 was shown by quantitative multiplex PCR in 17 of 30 (57%) of previously untested tumors. Immunoblot analyses revealed that 91% (20 of 22) retinoblastoma exhibited either a complete loss or a decrease of the intact form of CDH11 and 8 of 13 showed a prevalent band suggestive of the variant form. Copy number of WI-5835 for these samples correlated with CDH11 protein expression. CDH11 staining was evident in the inner nuclear layer in early mouse retinal development and in small transgenic murine SV40 large T antigen-induced retinoblastoma tumors, but advanced tumors frequently showed loss of CDH11 expression by reverse transcription-PCR, suggestive of a role for CDH11 in tumor progression or metastasis. CDH13 protein and mRNA were consistently expressed in all human and murine retinoblastoma compared with normal adult human retina. Our analyses implicate CDH11, but not CDH13, as a potential tumor suppressor gene in retinoblastoma.

Animals↗

PubMed2001

Study of microdeletions in the Y chromosome of infertile men with idiopathic oligo- or azoospermia.

PURPOSE: To determine the relationships between idiopathic oligo- or azoospermia and microdeletions of the Y chromosome. METHODS: Eighteen Y-linked sequence-tagged sites (STSs) in AZF (Azoospermia Factor) region were screened by means of multiplex PCR (Polymerase Chain Reaction) in 50 idiopathic infertile men, including 16 patients with azoospermia, 13 severe oligospermia, and 21 oligospermia. RESULTS: Microdeletions in the genomic DNA were observed in 8 of 50 cases, 3 with azoospermia, I severe oligospermia, and 4 oligospermia. Total deletion rate was 16.0% (8/50). The deletion regions were concentrated on AZFd and AZFc. CONCLUSIONS: Microdeletions of the Y chromosome are an important cause for idiopathic oligo- or azoospermia. Multiplex PCR is a useful technique for detecting the microdeletions. To avoid transmission to their offspring, patients with idiopathic oligo- or azoospermia should be screened for microdeletions of the Y chromosome before ICSI treatment for infertility.

Adult↗

PubMed2006

A new PCR-CGE (size and color) method for simultaneous detection of genetically modified maize events.

We present a novel multiplex PCR assay for simultaneous detection of multiple transgenic events in maize. Initially, five PCR primers pairs specific to events Bt11, GA21, MON810, and NK603, and Zea mays L. (alcohol dehydrogenase) were included. The event specificity was based on amplification of transgene/plant genome flanking regions, i.e., the same targets as for validated real-time PCR assays. These short and similarly sized amplicons were selected to achieve high and similar amplification efficiency for all targets; however, its unambiguous identification was a technical challenge. We achieved a clear distinction by a novel CGE approach that combined the identification by size and color (CGE-SC). In one single step, all five targets were amplified and specifically labeled with three different fluorescent dyes. The assay was specific and displayed an LOD of 0.1% of each genetically modified organism (GMO). Therefore, it was adequate to fulfill legal thresholds established, e.g., in the European Union. Our CGE-SC based strategy in combination with an adequate labeling design has the potential to simultaneously detect higher numbers of targets. As an example, we present the detection of up to eight targets in a single run. Multiplex PCR-CGE-SC only requires a conventional sequencer device and enables automation and high throughput. In addition, it proved to be transferable to a different laboratory. The number of authorized GMO events is rapidly growing; and the acreage of genetically modified (GM) varieties cultivated and commercialized worldwide is rapidly increasing. In this context, our multiplex PCR-CGE-SC can be suitable for screening GM contents in food.

DNA, Plant↗

PubMed1999

A convenient and efficient microsatellite-based assay for resolving parentages in dogs.

We present a practical and efficient parentage analysis test for dogs. The method makes use of ten polymorphic microsatellite markers combined in three multiplex-PCR reactions. All three multiplexes can be performed under the same cycling conditions and using a multiple-dye detection system the PCR-products can be pooled for electrophoresis. Amplifications in four different thermal cyclers produced easily interpretable results throughout demonstrating the repeatability of the assay. In order to evaluate the method's efficiency a total of 100 dogs from four different breeds were typed. Assuming one known parent, exclusion probabilities ranging from 99.34% to 99.93% were attained.

Animals↗

PubMed2004

High-throughput method for detecting genomic-deletion polymorphisms.

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.

DNA Probes↗

PubMed2004

Prenatal diagnosis of mos46,X,del(Y)(q11.2)/45,X by cytogenetic and molecular studies with multiplex STR analysis.

OBJECTIVES: To present the prenatal diagnosis of a fetus of mos46,X,del(Y)(q11.2)/45,X by cytogenetic and molecular analysis. CASE AND METHODS: A 35-year-old pregnant woman came to our hospital for amniocentesis, and fetal chromosomal aberrations with mos46,X, + mar/45,X were found. Fluorescence in situ hybridization revealed the existence of a Y centromere on the marker chromosome. Analysis with six pairs of short tandem repeat markers showed that the genomic DNA extracted from the uncultured amniotic fluid cells contained a deletion of Yq11.1-Yq11.2. Spermatogenesis loci of the Y chromosome were studied using four sets of multiplex PCR. The proximal two markers DYS271 and KALY were present and the other 16 distal markers were deleted. No deletion was noted in the Y chromosome of the father. RESULTS: Cytogenetic and molecular analyses revealed deletions of AZFb, d, and c regions on Yq11.2-Yqter in the fetal Y chromosome. Postmortem examination of the fetus showed a grossly normal male fetus with normal external genitalia and testes. CONCLUSION: The present report demonstrates that molecular analysis using polymorphic microsatellite markers and multiplex PCR is a useful complement to cytogenetic methods for the identification and the characterization of Y-chromosomal deletions.

Adult↗

PubMed2000

Evolution of chloramphenicol resistance, with emergence of cross-resistance to florfenicol, in bovine Salmonella Typhimurium strains implicates definitive phage type (DT) 104.

The prevalence of resistance to florfenicol, a phenicol drug newly introduced in veterinary therapy, was determined in 86 chloramphenicol-resistant Salmonella Typhimurium isolates from cattle collected during 1985-1995. All were highly resistant to chloramphenicol (MICs > or = 128 mg/L) and 38 were simultaneously resistant to florfenicol (MICs >16 mg/L) and to beta-lactam agents, spectinomycin, streptomycin, sulphonamides and tetracyclines. The isolates susceptible to florfenicol harboured the chloramphenicol acetyl transferase gene, cat of type I. All the florfenicol-resistant isolates harboured the floR resistance gene and the characteristic multiple resistance genetic locus, previously characterised in a S. Typhimurium DT104 strain and identified by a multiplex PCR. Plasmid profiles and ribotype patterns were determined for all the isolates. The florfenicol-resistant isolates were grouped into the same ribotyping pattern and presented similar plasmid profiles, whereas the florfenicol-susceptible isolates showed a wider genetic diversity that is usual for S. Typhimurium. Thus, the florfenicol-resistant isolates could represent a clonal cluster, closely related to, if not of DT104 phage type, which appeared in 1989 and is now predominant within chloramphenicol-resistant S. Typhimurium. The multiplex PCR provided a useful tool to survey further evolution of multiresistant S. Typhimurium strains.

Animals↗

PubMed2005

MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-chromosomal recessive disorders caused by mutations in the dystrophin gene. Using the novel multiplex ligation-dependent probe amplification (MLPA) method we performed retrospective and prospective analyses in a total of 193 individuals. Deletions or duplications were identified in 14 out of 90 families previously tested negative by multiplex PCR or FISH analysis. Partially incorrect results were subsequently identified in two families: the loss of exon 38 signal in one case was due to a p.Q1802X nonsense mutation, whilst in another patient an apparent deletion of exon 37 (coinciding with a duplication of exons 46-53) was caused by a p.R1735C polymorphism. In one case we found a complex rearrangement involving a duplication of two regions: dupEX45-48 and dupEX54-55. We conclude that MLPA is a highly sensitive and rapid alternative to multiplex PCR. It can be used on blood samples, chorionic villi and paraffin-embedded tissue. The ease of detection of duplications and the application for female carrier analysis are clearly the main advantages of the method. However, apparent single exon deletions detected by MLPA should be checked by an independent method. Complex rearrangements such as double mutations on the same allele are rare.

DNA Primers↗

PubMed1995

[Problems found in genetic diagnosis of DMD/BMD].

The method to understand the result of the multiplex PCR amplification of the dystrophin gene has been discussed. In dystrophin Kobe, a very small intra-exon deletion was identified in exon 19 when the amplified product was not detected in its corresponding position. Also, an insertion mutation was identified in exon 44 of the dystrophin gene named dystrophin Yakumo. In this case the amplified product from exon 44 was not localized to the its expected position, but a larger fragment was identified. In one case of BMD all exons were amplified by multiplex PCR. However, thorough examination disclosed the presence of point mutation at the last nucleotide of exon 13.

Dystrophin↗

PubMed2006

Rapid multiplexing and simultaneous detection of human spermatogenetic failure with a 12 lane microchip electrophoresis system.

For the amplification and ultrafast separation of the genetic markers and DNA sequences that are related to human male infertility, a multiplex PCR for amplifying three DNA sequence-tagged sites (STS) located on the human Y chromosome with possible roles in the spermatogenesis process has been designed and applied followed by separation on a microchip. First, the optimum T(m) degree for the three DNA markers was optimized and determined experimentally, and the three DNA STS were amplified. These three DNA markers were then separated on a 12-lane microchip electrophoresis system, which can analyze the DNA markers on 12 channels simultaneously. The combination of these two technologies, multiplex PCR and microchip electrophoresis, allows the analysis of 36 DNA markers (12x3) within only 180 s.

Base Sequence↗

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↗

PubMed2002

Presence of the RHD pseudogene and the hybrid RHD-CE-D(s) gene in Brazilians with the D-negative phenotype.

The molecular basis for RHD pseudogene or RHD Psi is a 37-bp insertion in exon 4 of RHD. This insertion, found in two-thirds of D-negative Africans, appears to introduce a stop codon at position 210. The hybrid RHD-CE-Ds, where the 3' end of exon 3 and exons 4 to 8 are derived from RHCE, is associated with the VS+V- phenotype, and leads to a D-negative phenotype in people of African origin. We determined whether Brazilian blood donors of heterogeneous ethnic origin had RHD Psi and RHD-CE-Ds. DNA from 206 blood donors were tested for RHD Psi by a multiplex PCR that detects RHD, RHD Psi and the C and c alleles of RHCE. The RHD genotype was determined by comparison of size of amplified products associated with the RHD gene in both intron 4 and exon 10/3'-UTR. VS was determined by amplification of exon 5 of RHCE, and sequencing of PCR products was used to analyze C733G (Leu245Val). Twenty-two (11%) of the 206 D-negative Brazilians studied had the RHD Psi, 5 (2%) had the RHD-CE-Ds hybrid gene associated with the VS+V- phenotype, and 179 (87%) entirely lacked RHD. As expected, RHD was deleted in all the 50 individuals of Caucasian descent. Among the 156 individuals of African descent, 22 (14%) had inactive RHD and 3% had the RHD-CE-Ds hybrid gene. These data confirm that the inclusion of two different multiplex PCR for RHD is essential to test the D-negative Brazilian population in order to avoid false-positive typing of polytransfused patients and fetuses.

Black People↗

PubMed2004

[A new member of CMY type cephalosporinase prevailing in Escherichia coli].

OBJECTIVE: To study the resistant phenotype and molecular biology character of plasmid mediated high AmpC-producing clinical isolates of Escherichia coli and to find new AmpC genotype. METHODS: The cefoxitin highly resistant clinical isolates of Escherichia coli were studied by K-B method, three-dimensional method, Isoelectric Focusing (IEF) and the MIC of these strains were examined by micro-dilution method. The conjugation experiment, multiplex PCR and DNA sequencing methods were used in further study. RESULTS: Above 719 strains studied, there are 6 isolates were showed as high AmpC-producing by three-dimensional method and IEF found they could produce a beta-Lactamase which PI was 8.9 and could be inhibited by cloxacillin but not by clavulnate. The strains were resistant to most of third generation cephalosporins, but were susceptible to cefepime, meropenem and imipenem. The experiment also showed that the gene which express this AmpC like beta-Lactamase could be transferable. Multiplex PCR indicated they belong to Citrobacter freundii family. Sequencing of corresponding DNA revealed 99% identities of the deduced amino acid sequence with CMY-2 and CMY-7 respectively. It is a new CMY type cephalosporinase. CONCLUSION: A new CMY type cephalosporinase has been found in clinical strains of Escherichia coli in our hospital. It was resistant to many antibiotics and its resistance could be transferred horizontally.

Bacterial Proteins↗

PubMed2005

Deletion and duplication screening in the DMD gene using MLPA.

We have designed a multiplex ligation-dependent probe amplification (MLPA) assay to simultaneously screen all 79 DMD gene exons for deletions and duplications in Duchenne and Becker muscular dystrophy (DMD/BMD) patients. We validated the assay by screening 123 unrelated patients from Serbia and Montenegro already screened using multiplex PCR. MLPA screening confirmed the presence of all previously detected deletions. In addition, we detected seven new deletions, nine duplications, one point mutation, and we were able to precisely determine the extent of all rearrangements. To facilitate MLPA-based screening in laboratories lacking specific equipment, we designed the assay such that it can also be performed using agarose gel analysis and ethidium bromide staining. The MLPA assay as described provides a simple and cheap method for deletion and duplication screening in DMD/BMD patients. The assay outperforms the Beggs and Chamberlain multiplex-PCR test, and should be considered as the method of choice for an initial DNA analysis of DMD/BMD patients.

Gene Deletion↗

PubMed2005

[Prenatal determination of fetal RHD in maternal plasma: two-years experience of routine clinical use].

OBJECTIVES: To evaluate the predictive value of RHD fetal genotype in maternal plasma of Rh D negative mothers after 10 weeks of gestation in a clinical use. MATERIAL AND METHOD: Prospective, comparative study between fetal RHD genotyping in maternal plasma, with amplification of exons 4,5,10 of the RHD gene, by real-time multiplex PCR, and Rh D serology at birth, in 218 pregnancy and their 223 babies, between November 2002 and 2004. RESULTS: Combining the amplification of three exons, the concordance rate of fetal Rh D genotyping in maternal plasma and baby phenotyping at delivery was 100%. Four women whose the babies were Rh D negative were positive for RHD exon 10 during pregnancy. This positivity was, in three cases, correlated with the presence of RHDpsi pseudogene and in last case, with a haplotype Cdes (r's). RHD genotyping was performed for five twin pregnancies. CONCLUSION: Multiplex PCR using maternal plasma provides perfect prenatal prediction of fetal RHD gene. These results confirm that this non invasive procedure is the preferred method for assessing Rh D fetal status in Rh negative mothers. Using this method for two years in routine practice has led us to modify our management scheme for sensitized Rh D-negative pregnant women.

Female↗

PubMed2006

[Azoospermia factor microdeletions in idiopathic azoospermia and severe oligozoospermia].

OBJECTIVE: To observe the relationship between microdeletions of AZF( azoospermia factor) on Y chromosome in male with idiopathic azoospermia and severe oligozoospermia. METHODS: Only patients with an apparently normal 46,XY karyotype and normal FSH, LH and T were included in this study. Multiplex PCR was used to detect the sequence-tagged sites( STS) as follows :sY84, sY86, sY127, sY134, sY152, sY153, sY254, sY255, and ZFX/Y was used as internal control gene. RESULTS: No microdeletion was detected in the control whereas 8 microdeletion cases existed in 67 idiopathic azoospermia and severe oligozoospermia, including 4 in AZFc, 2 in AZFa + AZFc, 1 in AZFc + AZFb, and 1 in AZFb. The prevalence rate of microdeletion was 11.94%, which was statistically different from the control. CONCLUSION: Microdeletions in the AZF regions on the long arm of the Y-chromosome are associated with idiopathic azoospermic and severely oligozoospermic men. Multiplex PCR was a rapid and reliable method for screening microdeletions of AZF.

Adult↗

PubMed2005

Antimicrobial resistance and class 1 integrons in pathogenic Escherichia coli from dairy farms.

The goal of this study was to assess the prevalence of antimicrobial resistance and class 1 integrons, including integron-associated genes, in 24 Escherichia coli isolates from dairy farms. Escherichia coli isolates (n = 14) from dairy cows with mastitis (ECDM), Shiga toxin-producing (STEC) O157:H7 from cull dairy cow fecal samples (n = 9) and bulk tank milk (n = 1) were evaluated for sensitivity to 19 antimicrobial agents used commonly in human and/or veterinary medicine. Multiplex PCR was used to determine presence of genes associated with class 1 integrons (intI1, qacEDelta1, and sulI1). Class 1 integrons were found only in eight of 10 isolates (one STEC O157:H7 and seven ECDM) that demonstrated antimicrobial resistance, and seven of these were resistant to two or more antimicrobial agents. Eight of 10 STEC O157:H7 and six of 14 ECDM were susceptible to all commonly used antibiotics. Five ECDM demonstrated multiple resistances to four or more antibiotics. Most of the 24 isolates examined exhibited resistance against sulfamethoxazole, followed by streptomycin and tetracycline. STEC O157:H7 strains had less prevalence of antibiotic resistance and integron carriage than ECDM. The multiplex PCR method developed for detection of intI1, qacEDelta1, and sulI1 can be used routinely for monitoring presence of these genes. Class 1 integrons were found in eight of 10 E. coli strains that demonstrated antimicrobial resistance; seven of these were resistant to two or more antibiotics. It appears that integrons played a role in the incidence of antimicrobial resistance of the strains used in this study.

Animals↗

PubMed2006

Detection and diversity of various Arcobacter species in Danish poultry.

The prevalence and diversity of different Arcobacter spp. in various poultry species in Denmark were investigated using cultural and multiplex PCR methods. A pool of three fresh droppings obtained at the production site from 70 broiler chicken flocks aged 4-5 weeks was examined. In addition, pools of 10 cloacal swabs taken at the abattoir prior to stunning from each of 15, and 37 duck and turkey flocks, respectively, were analyzed. Thirty fresh broiler chicken carcasses and 29 cloacal swabs from the respective viscera were also examined at the abattoir. Finally, 10 caecal and 10 cloacal swabs from ducks at the abattoir were analyzed individually. In total, 85 Arcobacter isolates were obtained. Of these 45, 20 and 7 were identified as Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively, using a multiplex PCR. Interestingly, some chicken isolates of A. butzleri showed urease activity, and 6 out of seven A. skirrowi isolates were unable to hydrolyse indoxyl acetate. All chicken carcasses examined were found positive for A. butzleri and/or A. cryaerophilus, whereas 21 (72%) of the 29 chicken cloacal swabs were positive for either A. butzleri (13) or A. cryaerophilus (9). Three (4.3%) out of 70 chicken flocks analyzed were positive only for A. cryaerophilus. Of the ten ducks examined individually, 7 carried A. skirrowii and/or A. cryaerophilus in their cloacae. None of the respective caecal samples were positive. Of the remaining 15 duck flocks, 11 (73%) were positive for A. cryaerophilus (7), A. butzleri (2) or A. skirrowii (2). Four (11%) of the 37 turkey flocks analyzed harboured either A. butzleri or A. cryaerophilus. The carriage rate of Arcobacter was higher in live ducks than those of live broiler chickens and turkeys in the present study. In addition, chicken carcasses slaughtered in Denmark were found to be contaminated with Arcobacter. The presence of Arcobacter spp. both on chicken carcasses and in poultry intestine may be of significance to human health.

Abattoirs↗