Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in‐frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.     

多重聚合酶链反应技术快速鉴定鲍曼不动杆菌

目的建立快速鉴定鲍曼不动杆菌菌株的方法。方法本研究建立多重PCR实验技术快速鉴定170株醋酸钙-鲍曼不动杆菌复合体以及对照组的其他菌属14株。结果138株菌的PCR产物扩增出2条条带,为鲍曼不动杆菌,另外32株只扩增出1条条带,为醋酸钙-鲍曼不动杆菌复合体的其他基因型,对照组的菌株没有扩增出条带。结论多重PCR技术的建立为快速鉴定鲍曼不动杆菌提供了一个快速而简便的方式。     

Exon Deletion Patterns of the Dystrophin Gene in 82 Vietnamese Duchenne/Becker Muscular Dystrophy Patients

Abstract

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited muscle diseases caused by mutations in the dystrophin gene. The reading frame rule explains the genotype-phenotype relationship in DMD/BMD. In Vietnam, extensive mutation analysis has never been conducted in DMD/BMD. Here, 152 Vietnamese muscular dystrophy patients were examined for dystrophin exon deletion by amplifying 19 deletion-prone exons and deletion ends were confirmed by dystrophin cDNA analysis if necessary. The result was that 82 (54%) patients were found to have exon deletions, thus confirming exact deletion ends. A further result was that 37 patterns of deletion were classified. Deletions of exons 45–50 and 49–52 were the most common patterns identified, numbering six cases each (7.3%). The reading frame rule explained the genotype-phenotype relationship, but not five (6.1%) DMD cases. Each of five patients had deletions of exons 11–27 in common. The applicability of the therapy producing semifunctional in frame mRNA in DMD by inducing skipping of a single exon was examined. Induction of exon 51 skipping was ranked at top priority, since 16 (27%) patients were predicted to have semifunctional mRNA skipping. Exons 45 and 53 were the next ranked, with 12 (20%) and 11 (18%) patients, respectively. The largest deletion database of the dystrophin gene, established in Vietnamese DMD/BMD patients, disclosed a strong indication for exon-skipping therapy.     

Cost Analysis of Multiplex PCR Testing for Diagnosing Respiratory Virus Infections

We performed a cost analysis study using decision tree modeling to determine whether the use of multiplex PCR testing for respiratory viruses (xTAG RVP test) is a more or less costly strategy than the status quo testing methods used for the diagnosis of respiratory virus infections in pediatric patients. The decision tree model was constructed by using four testing strategies for respiratory virus detection, viz., direct fluorescent-antibody staining (DFA) alone, DFA plus shell vial culture (SVC), the xTAG RVP test alone, or DFA plus the xTAG RVP test. A review of the charts of 661 pediatric patients was used to determine the length of hospital stay, the number of days in isolation, antibiotic usage, and all other medical procedures performed. The cost of hospitalization by diagnostic status was determined on the basis of the average cost per patient and the number of patients in each arm of the decision tree. The cost per case was the highest for DFA plus SVC at $3,914 (in Canadian dollars), and the lowest was for the xTAG RVP test alone at $3,623, while the costs of DFA alone ($3,911) and DFA plus RVP ($3,849) were intermediate. When all four diagnostic strategies were compared, the least costly strategy was the xTAG RVP test alone when the prevalence of infection was 11% or higher and DFA alone when the prevalence was under 11%. These data indicate a savings of $291 per case investigated if the strategy of using the xTAG RVP test alone was used to replace the status quo test of DFA plus SVC, resulting in a savings of $529,620 per year in direct costs for the four Hamilton, Ontario, Canada, hospitals on the basis of the testing of specimens from 1,820 pediatric inpatients. We conclude that the use of the xTAG RVP test is the least costly strategy for the diagnosis of respiratory virus infections in children and would generate a significant savings for hospitals.Clinical virology laboratories have historically used traditional methods, such as culture, direct fluorescent-antibody staining (DFA), and enzyme immunoassay, for the diagnosis of respiratory tract infections (3). DFA offers a rapid turnaround time for results but is labor-intensive and subjective and requires specific monoclonal antibodies and trained technologists. Both DFA and shell vial culture (SVC) are limited by the availability of monoclonal antibodies, precluding their use for the detection of newly discovered viruses. DFA has a low sensitivity for the detection of some viruses, especially adenovirus, and many laboratories reflex DFA-negative specimens into SVCs to improve the detection rates. For traditional methods such as DFA and SVC, turnaround times for results can be slow for laboratories handling large volumes of specimens. Rapid enzyme immunoassays have been used for the detection of influenza virus and respiratory syncytial virus (RSV), but these tests are only 50 to 70% sensitive (5, 15), which limits their use to specific point-of-care settings at times when the prevalence of infection is high.Over the past 10 years, nucleic acid amplification tests have been developed for an increasing number of respiratory viruses. Nucleic acid amplification tests, including PCR and nucleic acid-sequence based amplification, have shown enhanced sensitivity compared with the sensitivities of DFA and culture for the detection of a number of respiratory viruses (8). The emergence of five new respiratory viruses since 2000, including human metapneumovirus, the sudden acute respiratory syndrome-associated coronavirus, avian influenza virus H5N1, coronaviruses NL63 and HKU1, and human bocavirus, has presented new challenges for clinical laboratories. The absence of commercially available tests for the detection of these emerging viruses often leaves laboratories without the ability to diagnose these important virus infections. Multiplex PCR assays for the detection of multiple respiratory viruses have recently been introduced (for a review, see reference 8). These multiplex assays have heralded a new era in the molecular diagnostics of respiratory virus infections. Some of these tests are now commercially available and can detect up to 18 different respiratory viruses (6). A multiplex PCR test for respiratory viruses (the xTAG RVP test) is the first multiplex PCR to be cleared by the U.S. Food and Drug Administration and has been approved for use for the detection of 12 different respiratory viruses (16). The xTAG RVP test detects 30 to 40% more virus infections than DFA and culture, in part because it is more sensitive for the detection of traditional respiratory viruses, but it also detects nine additional viruses not detected by DFA and SVC (6, 7, 9, 10, 14). These newer multiplex tests are often costly, and the clinical and economic impacts of their implementation in routine hospital laboratories have not been evaluated. We therefore conducted a cost analysis study to compare the costs of the xTAG RVP test to those of conventional tests for the diagnosis of respiratory virus infections in hospitalized patients.     

Evaluation of Pneumococcal Serotyping by Multiplex PCR and Quellung Reactions

Screening of 1,750 pneumococcal isolates for common serotypes by PCR was followed by Quellung reaction analysis of PCR-negative isolates with a comparison to the conventional (Quellung reaction only) approach. PCR agreed with Quellung reaction results for 99% of isolates. The sequential PCR/Quellung reaction algorithm is accurate and more cost-effective than the conventional approach.     

Differential Detection of Five Mouse-Infecting Helicobacter Species by Multiplex PCR

Several species of helicobacter have been isolated from laboratory mice, including H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, which appear to be the most common. The most widely used published method for molecular detection of these agents is PCR amplification of a conserved region of 16S rRNA, but differential speciation requires restriction enzyme digestion of the amplicons. This study was undertaken to determine PCR conditions that would simultaneously and specifically identify each of the five common species without restriction enzyme analyses. First, we designed novel and specific PCR primers for H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, using sequences from the heterologous regions of 16S rRNA. Because of comigration of amplified products, we next identified P17, an H. bilis-specific protein; P25, an H. hepaticus-specific protein; and P30, an H. muridarum-specific protein by screening genomic DNA expression libraries of each species. Primers were designed from these three genes, plus newly designed, species-specific 16S rRNA primers for H. rodentium and H. typhlonius that could be utilized for a five-plex PCR. The sizes of the amplicons from H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius were 435, 705, 807, 191, and 122 bp, respectively, allowing simultaneous detection and effective discrimination among species.     

Analysis by Multiplex PCR of the Physical Status of Human Papillomavirus Type 16 DNA in Cervical Cancers

Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concomitant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression.     

Monitoring of Patients Supported by Extracorporeal Membrane Oxygenation for Systemic Infections by Broad-Range rRNA Gene PCR Amplification and Sequence Analysis

The rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative.     

Multiplex PCR To Diagnose Bloodstream Infections in Patients Admitted from the Emergency Department with Sepsis

Sepsis is caused by a heterogeneous group of infectious etiologies. Early diagnosis and the provision of appropriate antimicrobial therapy correlate with positive clinical outcomes. Current microbiological techniques are limited in their diagnostic capacities and timeliness. Multiplex PCR has the potential to rapidly identify bloodstream infections and fill this diagnostic gap. We identified patients from two large academic hospital emergency departments with suspected sepsis. The results of a multiplex PCR that could detect 25 bacterial and fungal pathogens were compared to those of blood culture. The results were analyzed with respect to the likelihood of infection, sepsis severity, the site of infection, and the effect of prior antibiotic therapy. We enrolled 306 subjects with suspected sepsis. Of these, 43 were later determined not to have infectious etiologies. Of the remaining 263 subjects, 70% had sepsis, 16% had severe sepsis, and 14% had septic shock. The majority had a definite infection (41.5%) or a probable infection (30.7%). Blood culture and PCR performed similarly with samples from patients with clinically defined infections (areas under the receiver operating characteristic curves, 0.64 and 0.60, respectively). However, blood culture identified more cases of septicemia than PCR among patients with an identified infectious etiology (66 and 46, respectively; P = 0.0004). The two tests performed similarly when the results were stratified by sepsis severity or infection site. Blood culture tended to detect infections more frequently among patients who had previously received antibiotics (P = 0.06). Conversely, PCR identified an additional 24 organisms that blood culture failed to detect. Real-time multiplex PCR has the potential to serve as an adjunct to conventional blood culture, adding diagnostic yield and shortening the time to pathogen identification.The emergency department (ED) often serves as the front line for the initial evaluation and management of patients with infections, with estimates indicating that there are more than 3.5 million visits to EDs annually (26). Community-acquired sepsis carries a large health care burden and accounts for a substantial proportion of these visits. Furthermore, recent studies suggest a rising burden of sepsis, with the annualized increase being about 8.7% (23). Many of these patients, estimated at 3 per 1,000 population (1), progress to severe sepsis. Despite improvements in the management of severe sepsis (15, 27), the case fatality rate ranges from 10% in children to 38.4% in the elderly (1). However, sepsis case fatality rates can be reduced by 33% to 77% with the early administration of adequate antibiotics (4, 10, 17, 18, 34). The antibiotics initially chosen are often broad and empirical, with modifications being made after microbiological diagnostic information becomes available. Blood culture remains the “gold standard” for the identification of bacterial and fungal bloodstream infections, although it is limited by high volume requirements to maximize its sensitivity and often prolonged incubation times. In an effort to address some of these limitations, many advances have been developed to improve the sensitivity and decrease the time to identification of the cause of bloodstream infections.Molecular amplification techniques have been developed to replace the incubation step in blood culture. Conserved regions of microbial genomes serve as targets for amplification, such as the rRNA genes and the 16S-23S interspace region (13, 25). The identification of this species-specific amplified DNA is coupled with one of a variety of detection schemes for the rapid real-time identification of pathogen DNA in the bloodstream. Many protocols that target individual species (8) or multiple pathogens within a particular clade have been developed, such as panbacterial and panfungal assays (6, 12, 29). Within the past year, the simultaneous detection of up to 25 common bacterial and fungal pathogens by the use of a multiplex real-time PCR format has been reported (19, 21, 22, 30, 32, 33). However, many of those studies defined operating characteristics on the basis of repeated sampling of a small but highly morbid patient population, which can lead to spectrum and context biases. We hypothesized that PCR and blood culture can identify a partially overlapping group of bloodstream infections, making PCR a useful adjunct to blood culture. This study aims to define the operating characteristics of the SeptiFast multiplex real-time PCR assay compared to those of conventional blood culture for the diagnosis of bloodstream infections in patients with suspected community-acquired sepsis presenting to the ED.     

Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs), mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs) in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA) a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.     

dystrophin基因突变研究进展

Duchenne/Beckermuscu lar dystroph ies(DMD/BMD)是人类常见的X染色体连锁隐性遗传的神经-肌肉系统疾病,发病率高,至今尚无有效的治疗方法。DMD和BMD是等位基因异质性疾病,其候选基因dystroph in突变率高,有5′端和中央两个缺失热点区域,通过筛查缺失热点区域可以检出98%的缺失。DMD/BMD与dystroph in基因突变关系复杂,dystroph in基因突变导致DMD/BMD不仅与基因突变是否破坏了开放阅读框有关,而且与dystroph in蛋白受累的功能区域相关,并涉及“外显子跨越转录”和“阅读框重建”等保护性调节机制。     

High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis

Salmonella enterica is a leading cause of food-borne illness worldwide and is also a major cause of morbidity and mortality in domestic and wild animals. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and nonhuman serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR while incorporating a 6-carboxyfluorescein-labeled universal primer to fluorescently label all amplicons. The fluorescently labeled PCR products were separated using capillary electrophoresis, and a Salmonella multiplex assay for rapid typing (SMART) code was generated for each isolate, based upon the presence or absence of PCR products generated from each target gene. Seven hundred fifty-one blind clinical isolates of Salmonella from Washington State, collected in 2007 and previously serotyped via antisera, were screened with the assay. A total of 89.6% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously determined serotype, while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to that of conventional serotyping, but at a fraction of both the cost and time required per test.Infection with Salmonella in humans and animals primarily causes self-limiting gastrointestinal infections with mild to moderate symptoms, including fever, abdominal cramps, and diarrhea (26). More severe clinical outcomes, including death, may occur in cases of bacteremia or enteric fever (typhoid), which is often characterized by severe headaches and high fever but no diarrhea (5). Humans with typhoid may also become asymptomatic carriers who are capable of spreading the disease either through direct human contact or via fecal contamination of food. However, the most common mode of transmission for the majority of Salmonella infections in humans occurs through the consumption of contaminated foodstuffs and water. Recent estimates from the Food-Borne Diseases Active Surveillance Network of the Centers for Disease Control and Prevention (CDC) suggest that approximately 1.4 million cases of salmonellosis occur annually in the United States, resulting in 15,000 hospitalizations and 400 deaths (47). Furthermore, estimates of the economic impact of Salmonella infections in the United States suggest that annual expenditures due to lost productivity and medical care may be up to $2.3 billion (22).Salmonella bongori and S. enterica are the two species that comprise the genus Salmonella. S. enterica is further divided into six subspecies, namely, enterica (I), salamae (II), arizonae 5 (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI). S. enterica subsp. enterica strains are of the greatest clinical relevance and are typically isolated from humans and warm-blooded animals. Strains belonging to one of the other five S. enterica subspecies and S. bongori are associated with environmental or reptilian sources (10, 23a). Serologic classification of Salmonella strains based upon properties of various surface polysaccharide (O) and flagellar (H) antigens is the reference method for epidemiologic surveillance. This method involves the characterization of over 150 unique O and H antigens to produce an antigenic formula that can be scored using the Kauffman-White scheme to determine a serovar for an isolate (7, 23a). Currently, serotyping classifies over 2,500 serovars of Salmonella, of which over 1,400 belong to S. enterica subsp. enterica (7, 12). Although serotyping using the Kauffman-White scheme remains the standard for serovar determination through its longstanding and widespread use, it is not without significant deficiencies. Aside from being labor-intensive and expensive, serotyping is also time-consuming to perform, often taking three or more days after receipt of a specimen for a highly trained laboratory technician to produce a result. Incomplete or incorrect serologic classification may occur due to atypical expression of an isolate''s surface O or H antigen as in the case of mucoid strains in which the O antigen is obscured or for nonmotile and/or monophasic isolates for which only one flagellar phase antigen can be determined. Recent comparative genomic studies of common clinical serotypes have also revealed evidence of a high level of intraserovar variation among isolates of some serovars (16, 29, 38). In other cases, some serovars have been shown to be highly similar genetically, which suggests that a “genovar” classification be adopted, based upon genetic relatedness. It is also proposed that genovar classification may be more appropriate than serovar classification (4, 39).The deficiencies of conventional serotyping combined with the wealth of genomic information now available for Salmonella have led to the development of alternative molecular strategies to replace or complement conventional serotyping. A number of recent strategies have employed PCR-based approaches to determine different O and H antigens as a means to replace serologic identification of these antigens (14, 20, 25, 32). Others have proposed alternative strategies examining genetic differences as a means of identifying serovars, including ribotyping (17), pulsed-field gel electrophoresis (PFGE) (28), multiplex PCR (3, 4, 29), IS200 analysis (18, 46), random amplification of DNA polymorphisms (45), and DNA microarray analysis (38, 42).For this study, a rapid, high-throughput multiplex PCR-based method was developed that allows for discrimination of the majority of common serotypes that are reported in the United States based upon their genetic differences. The gene targets are present in some serotypes but not others, as demonstrated by previous comparative genomic studies (19, 35, 38, 39). Primers for all 16 gene targets were designed for use in a single multiplex PCR, thus allowing for a reduction in costs, a decrease in turnaround time per test, and a lower rate of laboratory error. Additionally, the scoring of resulting amplicons was adapted for high-throughput testing by fluorescently labeling PCR products and employing capillary electrophoresis for separation and analysis. Aside from offering high sensitivity and accurate sizing of the PCR amplicons, DNA sequencing equipment is robust and already common to a large number of public health laboratories. The use of this typing method, especially in conjunction with serogrouping or PFGE, allows for serovar determination of Salmonella isolates at a level comparable to that of conventional serotyping, with considerable time and cost savings.     

Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10² CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Fabry disease: Detection of gene rearrangements in the human α-Galactosidase A Gene by Multiplex PCR Amplification

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from lesions in the α-galaciosidase A gene leading to deficient or absent activity of the lysosomal hydrolase. To facilitate the detection of rearrangements in this 14-kb gene, a method was developed for the PCR amplification of all seven exons from genomic DNA in a single multiplex reaction. The entire coding region and all the intron/exon boundaries were amplified as four products. Application of this method permitted the detection of all five partial deletions previously identified by Southern analysis. This rapid method can be used to identify gene rearrangements in affected hemizygotes and determine heterozygosity for at risk females in families with Fabry disease. © 1993 Wiley-Liss, Inc.     

Multiplex PCR for typing strains of Toxoplasma gondii

A multiplex PCR assay was designed for multilocus strain typing of Toxoplasma gondii based on length polymorphism of five microsatellite markers. Eight T. gondii strains already sequenced at these five markers were used as control isolates. This method is simple, rapid, reproducible, and adapted to a large set of isolates.     

Multiplex PCR: optimization and application in diagnostic virology

PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.     

Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the alpha, beta, and gamma intimin variants.     

Muscle Plasma Membrane Changes in Dystrophin Gene Exon 52 Knockout Mouse

Changes in muscle plasma membranes in mice lacking exon 52 of the dystrophin gene (mdx52 mouse) were studied using the freeze-fracture technique. The extensor digitorum longus (EDL) muscle plasma membrane of the mdx52 mouse at 8 weeks of age showed significantly increased caveola density (p < 0.05 by two-tailed t-test) and significantly decreased densities of intramembranous particles (IMPs), orthogonal arrays (OAs) and orthogonal array subunit particles (OASPs) (p < 0.05 by two-tailed t-test, p < 0.01 by Wilcoxon rank-sum test, p < 0.05 by two-tailed t-test, respectively) on the protoplasmic face when compared with those of control EDL muscles. These changes are more similar to those seen in DMD than those in the mdx mouse at the same age as reported previously. Thus, the gene abnormality in the different exon of the mouse dystrophin gene seems to induce somewhat different changes in the muscle plasma membrane.