Exon deletions and duplications in BRCA1 detected by semiquantitative PCR

rearrangements have recently been identified in the BRCA1 gene. Inclusion of a method for identifying such rearrangements should now be a prerequisite for providing a comprehensive mutation detection strategy. We have developed a semiquantitative PCR-based fluorescent assay for the detection of previously identified deletions. This method avoids the need for long PCR or Southern blotting and is suitable for large-scale epidemiological studies. The assay was used to screen 44 high-risk families within the U.K. Yorkshire Health Region. No deletions were detected, but five cases (11%) with an apparent duplication of exon 13 in BRCA1 were identified. The presence of this mutation was confirmed by long PCR. Further developments include extending the assay to include all exons of BRCA1.     

Locations of crossover breakpoints within the CMT1A-REP repeat in Japanese patients with CMT1A and HNPP

We have used human β2 and β4 cDNA probes to map the genes encoding two isoforms of the regulatory β subunit of voltage-activated Ca²⁺ channels, viz. CACNB2 (β2) and CACNB4 (β4), to human chromosomes 10p12 and 2q22-q23, respectively, by fluorescence in situ hybridization. The gene encoding the β2 protein, first described as a Lambert-Eaton myasthenic syndrome (LEMS) antigen in humans, is found close to a region that undergoes chromosome rearrangements in small cell lung cancer, which occurs in association with LEMS. CACNB2 (β2) and CACNB4 (β4) genes are members of the ion-channel gene superfamily and it should now be possible to examine their loci by linkage analysis of ion-channel-related disorders. To date, no such disease-related gene has been assigned to 10p12 and 2q22-q23. Received: 5 February 1997 / Accepted: 4 April 1997     

多重实时荧光PCR相对定量法快速诊断唐氏综合征

为了建立一种基于多重实时荧光相对定量PCR技术并应用之于唐氏综合征分子诊断, 选择21号染色体上唐氏综合征特异区域基因片段(DSCR3)为目的基因, 以12号染色体上的磷酸甘油醛脱氢酶基因(GAPDH)为参照基因, 设计合成两对引物以及分别以不同荧光标记的TaqMan探针, 在同一个反应管中进行扩增。以相对定量指标△CT值区分唐氏综合征患者与正常人。采用EB 病毒转化技术, 把唐氏综合征患者外周血B 淋巴细胞转化成永生淋巴母细胞系作为标准品。通过优化反应条件, 使得目的基因和参照基因的扩增效率基本一致, 接近100%, 模板浓度在3～300 ng/μL范围内, △CT值的变异系数小于15%, 浓度在30 ng/μL时, 变异系数最小(＜10%), 以该浓度的DNA作为模板进行批内和批间实验的△CT值重复性好, 变异系数分别为9.8%和13.3%。运用建立的方法检测20例唐氏综合征患者的血标本和30例正常人的血标本, 正常人△CT值范围是-1.90～-1.30, 患者的△CT值范围是-2.95～-2.15, 两组之间无交叉重叠, 有明显差异(P＜0.001)。唐氏综合征患者永生细胞系建系成功 ,染色体核型和DNA 分析表明建系前后遗传是稳定的。因此, 实时荧光定量PCR比较△CT值的相对定量法快速诊断唐氏综合征是可行的。     

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis

Summary Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.     

Quantitative multiplex real-time PCR for detection of PLP1 gene duplications in Pelizaeus-Merzbacher patients

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive disorder of central nervous system (CNS) myelination typically affecting males. A genomic duplication of variable size at Xq22.2, containing the entire proteolipid protein 1 gene (PLP1), is responsible for approximately 60-70% of PMD cases. The aim of this study was to develop a rapid and robust method for determination of PLP1 gene dosage. We optimized two multiplex real-time quantitative PCR (Q-PCR) assays targeting exons 3 and 6 of the PLP1 gene, and then validated these assays by retrospective analysis of a set of genomic DNAs from 67 previously tested patients and 43 normal controls. Samples were analyzed in multiplex PCR reactions using TaqMan chemistry and the ABI Prism 7000 Sequence Detection System. PLP1 dosage was determined by the relative quantitative comparative threshold cycle method (DeltaDeltaCt) using the human serum albumin gene as the endogenous reference gene. Three clearly non-overlapping ranges of results, corresponding to the presence of one, two, or three PLP1 copies, were detected in both assays. The results were completely concordant with gender and previous PLP1 gene dosage testing based on quantitative fluorescent multiplex PCR and analysis of a dinucleotide polymorphism in the first intron of the PLP1 gene. We conclude that multiplex real-time Q-PCR represents a fast and reliable tool for PLP1 duplication testing in PMD families.     

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.     

Rapid categorization of pathogenic Escherichia coli by multiplex PCR

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.     

Rapid detection of Salmonella enterica serovars by multiplex PCR

A multiplex PCR based assay was developed for the identification of the genus Salmonella. Five sets of primers from different genomic sequences such as fimA, himA, hns, invA and hto genes were selected for the identification of serogroups of Salmonella enterica such as S. Typhi, S. ParatyphiA, S. Typhimurium, S. Enteritidis and S. Weltevreden. The selected primers amplified products with the sizes of 85, 123, 152, 275 and 496 bp, respectively, for the genus Salmonella. This assay was found to be highly sensitive, as it could detect 5 cells of Salmonella and 1,000 fg of genomic DNA. Amplification of DNA extracted from other genera viz. V. cholerae and E. coli yielded negative results. This assay provides specific and reliable results and allows for the cost–effective detection of Salmonella in one reaction tube in mixed bacterial communities.     

Comprehensive detection of genomic duplications and deletions in the DMD gene,by use of multiplex amplifiable probe hybridization

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening.     

The viral bronchiolites diagnosis in children by PCR multiplex

The aim of the study was to determine the etiology of the viral bronchiolites in children by using direct immunofluorescence test and 3 RT-PCR Multiplex (S.Bellau-Pujol) The study was performed on 122 nasal inspirations collected from 3 weeks-6 month old children hospitalizated in the pediatrics service of CH Rouen. The results were that the majority (53%) of bronchiolites in children had like etiology RSV and a lot of these infections had double viral etiology (26% RSV+ Rhinovirus; 2,7% RSV+HMPV and 1% RSV+Coronavirus 229E). An important viral factor which gives bronchiolitis in children is HMPV (11%). We also find respiratory infections with triple viral etiology: RSV+Influenza A virus + Rhinovirus.     

多重PCR-DHPLC快速检测食品中产志贺毒素大肠杆菌

应用多重PCR(motiplex PCR)结合变性高效液相色谱技术(denaturing high-performanceliquid chromatography,DHPLC)建立了快速检测食品中产志贺毒素大肠杆菌O111和O157的方法.以基因wzxO111、rfbEO157为靶基因,建立多重PCR-DHPLC方法,进行特异性和灵敏度测试,同时进行RT-PCR检测比较灵敏度.该方法具有良好特异性,可以一次PCR扩增同时检测O111、O157;灵敏度达到25 CFU/mL.129份牛肉样品中检出1例O111,3例O157阳性;74份鸡肉样品中检测出O111、O157阳性各1例,67份蔬菜样品中未检测到O111、O157.本文建立O111、O157多重PCR-DHPLC检测方法,操作简便,特异性强,适用于产志贺毒素大肠杆菌筛选检测.     

Characterization of 20 microsatellite loci by multiplex PCR in swimming crab, Portunus trituberculatus

The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura), is one of the most important fishery resources in East Asia. The aim of this work is to develop microsatellite multiplex PCR systems for 20 microsatellites which were isolated from a microsatellite-enriched genomic library and to explore the genetic diversity of P. trituberculatus captured in the coastal regions of the Yellow Sea in Korea. We prepared four multiplex PCR systems (two hexplex and tetraplex systems each) which could be used for the genotyping of 20 markers. Most markers in this system are highly informative with mean polymorphism information contents and observed heterozygosity of 0.913 and 0.930. The total number of alleles observed in this study was 724, and the mean allelic number per locus was 36.2. Significant deviations from the Hardy–Weinberg equilibrium (HWE) were observed at six loci in the Wonsando Island population, but all the loci deviated from the HWE in the Jeonjangpo population. The genetic differentiation between the two populations and the phylogenetic tree constructed by the unweighted pair group method with the arithmetic average suggested that there is no significant genetic difference between Wonsando Island and Jeonjangpo populations. The prepared multiplex PCR systems and population genetic data will be helpful to study phylogeographic analysis as well as to prepare strategies of stock management and aquaculture of P. trituberculatus.     

Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR

Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise.In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities. Received: 23 January 1996 / Revised: 21 February 1996     

Detection of deletions by the amplification of exons (multiplex PCR) in Duchenne muscular dystrophy

The polymerase chain reaction is a new powerful method for in vitro cloning of specific regions of DNA. The use of the heat-stable DNA polymerase made the reaction amenable to automation. This method greatly facilitates the detection of mutations which are responsible for Duchenne muscular dystrophy, via DNA amplification of multiple deletions prone exons from the DMD gene.     

Fin duplications and deletions induced by disruption of retinoic acid signaling

 Retinoic acid (RA), a derivative of vitamin A, plays a critical role as a signaling molecule in axial patterning of vertebrates. Here we report that RA exposure of zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) embryos during gastrulation results in homeotic duplications of the pectoral fins in up to 94% of fish. We have observed three to four pairs of fins in an individual fish. Although some duplications are partial, many represent complete axial duplications of the pectoral girdle and fin and include coracoscapulae, proximal radials, and dermal fin elements. Fin duplications are observed only at a defined dose of RA. Inhibition of RA synthesis by exposure to citral during a narrow developmental window leads to fish which lack pectoral fins but can be rescued by addition of exogenous RA, suggesting that RA signaling is critical to fin specification during early development. The ability to consistently induce multiple fins in a large number of vertebrate embryos should contribute to the understanding of genetic regulation of the normal positioning of limbs during embryogenesis. Received: 30 August 1997 / Accepted: 6 December 1997     

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.     

多重PCR快速检测化妆品中三种致病菌的研究

利用多重PCR技术建立快速检测化妆品中三种致病菌的方法。根据已报道的大肠杆菌phoA基因、铜绿假单胞菌外膜蛋白基因oprL和金黄色葡萄球菌特异性序列SmaI选择特异性引物,对人工染菌化妆品进行多重PCR检测。结果显示,三种致病菌的基因组DNA均可与各自引物特异性结合,扩增产物大小分别为622 bp、504 bp和426 bp。该方法用于人工污染的化妆品中,大肠杆菌的检出限浓度为103 CFU/mL,铜绿假单胞菌和金黄色葡萄球菌的检出限浓度为105 CFU/mL。作者建立的多重PCR方法可同时快速、特异地对化妆品中三种致病菌进行检测,在化妆品行业具有较大的应用价值。