Single PCR Assay Research Articles

Prevalence and distribution ofEimeriaspecies in broiler chicken farms of different capacities

We conducted a survey in broiler farms from Romania to establish prevalence and distribution of Eimeria species using single PCR assay. We found Eimeria spp. in 21 (91%) out of 23 flocks, and in 11 (92%) out of 12 farms. Four species of Eimeria were identified: E. acervulina (21/23; 91%), E. tenella (14/23; 61%), E. maxima (5/23; 22%) and E. praecox (3/23; 13%). Infection with a single species (E. acervulina) was detected in 6 (26%) infected flocks originated from large farms. Mixed infections were found in 15 (65%) flocks and the most prevalent combination was E. acervulina + E. tenella (8/23; 35%). Four flocks (17%) harboured mixed infection with E. acervulina + E. tenella + E. maxima. E. acervulina was significantly more prevalent in flocks that received ionophores as anticoccidial feed additives.

Occurrence of Beta2 toxigenic Clostridium perfringens isolates with different toxin types in Iran

Clostridium perfringens is an important cause of enteric diseases in both human and animals. The bacteria produce several toxins which play key roles in the pathogenesis of diseases and are classified into five toxin types, on the basis of the differential production of Alpha, Beta, Epsilon and Iota toxins. In this study a single PCR assay was developed and used for detection of cpb2 gene to identify the Beta2 harboring isolates among different types of C. perfringens isolated from animal enteric diseases in Iran. It was found that cpb2 presents among C. perfringens isolates types A, B, C and D with 54.5% (6/11), 62% (13/21), 42.8% (6/14), 69.25% (9/13), respectively. Totally 34 of 59 (56.7%) isolates screened by PCR were cpb2positive. This is the first report of cpb2 positive isolates of C. perfringens causing enteric diseases of animals in Iran. Further studies to demonstrate the exact role of Beta2 toxin in pathogenesis of the bacterium is suggested.

Primer1: Primer Design Web Service for Tetra-Primer ARMS-PCR

Tetra-primer ARMS-PCR is used extensively as a low cost, single PCR assay requiring no post-PCR manipulation. The design of successful primers depends on a number of variables such as melting temperatures, GC content, complementarity and selection of mismatch bases. The optimal selection of primers can be achieved in an automated way using a program which evaluates candidate primers for a given sequence. The Primer1 software was developed originally for use in the context of restriction fragment length polymorphism analysis using gel electrophoresis. However, recent applications have been more diverse, reviewed here, and we present an overview of the Primer1 software for primer design and web-service. We have updated the Primer1 program, and provide more complete details of the implementation. We also provide test data and output. The program is now available on a new, efficient, LAMP web service for users at: http://primer1.soton.ac.uk/primer1.html

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle.

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Bovine tuberculosis in South Darfur State, Sudan: an abattoir study based on microscopy and molecular detection methods

Bovine tuberculosis (BTB) is a widespread zoonosis in developing countries but has received little attention in many sub-Saharan African countries including Sudan and particularly in some parts such as Darfur states. This study aimed to detect bovine tuberculosis among caseous materials of cattle slaughtered in abattoirs in South Darfur State, Sudan by using microscopic and PCR-based methods. The study was a cross-sectional abattoir-based study which examined a total of 6,680 bovine carcasses for caseous lesions in South Darfur State between 2007 and 2009. Collected specimens were examined for the presence of acid-fast bacilli (AFB) by using microscopic and culture techniques. Isolated mycobacteria were identified by selected conventional cultural and biochemical tests in comparison to a single tube multiplex PCR (m-PCR) assay which detect Mycobacterium bovis-specific 168-bp amplicons. Of the total 6,680 slaughtered cattle examined in South Darfur, 400 (6 %) showed caseations restricted to lymph nodes (86.8 %) or generalized (13.2 %). Bovine tuberculosis was diagnosed in 12 (0.18 %), bovine farcy in 59 (0.88 %), unidentified mycobacteria in 6 (0.09 %), and missed or contaminated cultures in 7 (0.1 %). Out of 18 cultures with nonbranching acid-fast rods, 12 amplified unique 168-bp sequence specific for M. bovis and subsequently confirmed as M. bovis. With the exception of the reference M. tuberculosis strains, none of the remaining AFB amplified the 337-bp amplicon specific for M. tuberculosis. It could be concluded that bovine tuberculosis is prevalent among cattle in South Darfur representing 4.5 % from all slaughtered cattle with caseous lesions. The study sustains microscopy as a useful and accessible technique for detecting AFB. m-PCR assay proved to be valuable for confirmation of BTB and its differentiation from other related mycobacteriosis, notably bovine farcy.

Prevalence and risk factor analysis of Haemobartonella felis in cats using direct blood smear and PCR assay

The present case–control study was conducted to determine the prevalence of Haemobartonella felis, study the role of its risk factors and compare the different diagnostic tests. In order to achieve these goals, direct blood smear and single PCR assay were performed on blood samples from 90 cats (45 healthy controls and 45 sick cats). In addition, age, sex, breed, outdoor access, castration and density of living area were recorded in each group, and haematological analysis was performed on all blood samples. A total of 50 and 72.2 % of cats were H. felis positive in direct blood smear and PCR, respectively. The results showed a significant difference between control and case groups regarding the quantitative variables including fever and dehydration in direct blood smear, and tachycardia, icterus, fever and dehydration in PCR (P < 0.05). Castration, sex, breed, access to the outside (lifestyle) and density of cat population did not significantly affect test results. The results indicate that interactive effects of various factors (risk factors of infection) can diminish the individual specific effect of each.

One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB, gyrB, and ycfQ. Simultaneous gene scanning of fljB, gyrB, and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S. Dublin isolates, an additional single PCR assay was developed for specific identification of S. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.

Rapid detection of carbapenemase genes by multiplex real-time PCR

To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

New tools for quantifying HIV-1 reservoirs: plasma RNA single copy assays and beyond.

Quantification of plasma HIV-1 RNA below the limit of FDA-approved assays by a single copy quantitative PCR assays (SCA) has provided significant insights into HIV-1 persistence despite potent antiretroviral therapy as well as a means to assess the impact of therapeutic strategies, such as treatment intensification, on residual viremia. In this review, we discuss insights gained from plasma HIV-1 RNA SCA and highlight the need for additional assays to characterize better the cellular and tissue reservoirs of HIV-1. Accurate, reproducible, and sensitive assays to quantify HIV-1 reservoirs, before and after therapeutic interventions, are essential tools in the quest for a cure of HIV-1 infection.

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.

Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: Results in clinical benefits and cost saving

Introduction Among negative RhD mothers it is essential to know the fetal RhD status in order to avoid the possibility of hemolytic disease of the newborn. In this regard, the detection of fetal DNA in maternal plasma might become a new diagnostic tool. In the current study, we have evaluated the standardization of a Multiplex-PCR targeted towards two exons of the RHD and one SRY gene to monitor RhD negative women. The current study addresses questions concerning feasibility and applicability of this approach into the clinical practice. Materials and methods Both single and multiplex real-time PCRs targeting RHD exons 5 and 7 and SRY were applied for the detection of fetal-specific RHD sequences and sex in maternal plasma. A large cohort of 2127 women was studied between 10 and 28 weeks of pregnancy. 134 of them were used for single TaqMan PCR studies and 1993 were evaluated using Multiplex TaqMan PCR studies. All of them were serologically typed as RhD negative according to Spanish guidelines. Single and multiplex real-time PCR results were compared with postnatal serology and sex identification. Results There was a 100% concordance between results obtained with single and multiplex real-time PCR assays. At present, 1012 of the 1993 pregnant women studied gave birth and the results of RHD status obtained with the multiplex TaqMan PCR assay were confirmed postpartum by serological methods showing that sensitivity, specificity, and accuracy of the multiplex assay were 100, 98.6, and 99.3%, respectively. This procedure improved the speed of the assay, avoided over-treatment among RhD negative pregnant women bearing RhD negative fetus, and reduced the requirements for clinical and biological monitoring, resulting in a clinical benefit and cost saving. Conclusions The routine determination of fetal RHD status and SRY in maternal plasma, using multiplex real-time PCR, is feasible. The use of multiplex real-time PCR allows improving the response of the laboratory, saving time and reagent costs, opening the door to a complete automatization of the process.

Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus from clinical specimens

Type B influenza virus is one of the major epidemic strains and responsible for considerable mortality and morbidity. Rapidly and accurately identifying different influenza B virus lineages, i.e., B/Yamagata (B/Y) and B/Victoria (B/V), is desirable during the flu season. However, the available rapid techniques lack sensitivity, and the usual methods for identifying influenza viruses require expansion of virus in tissue culture or embryonated hen's eggs. Thus, we developed several sets of primer pairs that were able to detect and distinguish B/Y and B/V in a single real-time PCR assay. Used in conjunction with two sets of specific primers that exhibited purine at 3' end of at least one primer targeting on HA gene of B/Y and B/V lineages allows us to accurately identify approximately 10(2) copies per microliter for B/Y and B/V with intra- and inter-assay coefficient of variation (CV) <4%. When it was used to test 17,765 throat swab specimens obtained in the 2006-2010 influenza surveillance season, this method was comparable to hemagglutination inhibition assay in detection, typing and subtyping of influenza viruses with 100% true-negative (specificity) and 100% true-positive (sensitivity). Taken together, this method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination for WHO decisions on vaccine composition.

Trichothecene genotypes and chemotypes in Fusarium graminearum complex strains isolated from maize fields of northwest Argentina

Members of the Fusarium graminearum species complex (Fg complex) cause Gibberella ear rot in maize from northwest Argentina. The potential of these pathogens to contaminate maize grains with type B trichothecenes is a health risk for both humans and animals. We evaluated the reliability of multiplex PCR assays based on TRI3 and TRI12 genes, and single PCR assays based on TRI7 and TRI13 genes to infer trichothecene chemotypes of 112 strains of Fg complex collected from northwest Argentina, checking trichothecene production by chemical analysis. Single and multiplex PCR assays indicated that strains belonging to F. meridionale (87/112) had a NIV genotype. The remainder strains (25/112), which belonged to F. boothii, had a DON genotype (based on single PCR assays) or 15ADON genotype (based on multiplex PCR assays). No strains tested were incorrectly diagnosed with a DON/NIV genotype. Chemical analysis indicated that the F. meridionale strains were NIV producers only (44/87), major NIV producers with unexpected high DON/NIV ratios (36/87), or unexpected major DON producers with minor NIV production (7/87). Strains with atypical DON/NIV production seem to be new phenotypes under a putative NIV genotype, since PCR assays do not provide evidences of a new trichothecene genotype. DON production and absence of its acetylated forms were shown for strains of F. boothii. The inconsistencies between genetic and chemical data highlight the risk of inferring the trichothecenes potentially contaminating food and feedstuffs based only on PCR assays. This study confirms for the first time that strains of Fg complex from maize of northwest Argentina are DON and NIV producers. In addition, dominance of NIV producers in the Fg complex population isolated from maize is unprecedented in Argentina, and of significant concern to food safety and animal production.

Reliability and Short-Term Intra-Individual Variability of Telomere Length Measurement Using Monochrome Multiplexing Quantitative PCR

BackgroundStudies examining the association between telomere length and cancer risk have often relied on measurement of telomere length from a single blood draw using a real-time PCR technique. We examined the reliability of telomere length measurement using sequential samples collected over a 9-month period.Methods and FindingsRelative telomere length in peripheral blood was estimated using a single tube monochrome multiplex quantitative PCR assay in blood DNA samples from 27 non-pregnant adult women (aged 35 to 74 years) collected in 7 visits over a 9-month period. A linear mixed model was used to estimate the components of variance for telomere length measurements attributed to variation among women and variation between time points within women. Mean telomere length measurement at any single visit was not significantly different from the average of 7 visits. Plates had a significant systematic influence on telomere length measurements, although measurements between different plates were highly correlated. After controlling for plate effects, 64% of the remaining variance was estimated to be accounted for by variance due to subject. Variance explained by time of visit within a subject was minor, contributing 5% of the remaining variance.ConclusionOur data demonstrate good short-term reliability of telomere length measurement using blood from a single draw. However, the existence of technical variability, particularly plate effects, reinforces the need for technical replicates and balancing of case and control samples across plates.

P3-S1.41 Coinfection of Neisseria gonorrhoeae and Chlamydia trachomatis in symptomatic and asymptomatic women in India: implications in reproductive health

BackgroundDespite the recent advances in diagnosis, Neisseraia gonorrhoeae (NG) and Chlamydia trachomatis (CT) remain leading cause of STDs worldwide and account for STI's morbidity leading to acquisition of HIV and...

Abstract 4161: Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine

Abstract Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary Matrix Metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine. Methods: Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. These LNA suppress wild-type DNA amplification. To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform. Results: To validate the new the multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats. Conclusions: By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4161. doi:10.1158/1538-7445.AM2011-4161

Development of a real-time multiplex PCR assay for the detection of FGFR3 mutations in urine.

271 Background: We have recently reported the development of a Multi-Analyte Diagnostic Readout (MADR) non-invasive assay using urinary matrix metalloproteinases (MMPs) and FGFR3 as triage monitors in high-risk bladder cancer populations. This concept combines the marker performance characteristics of protein and DNA biomarkers into one assay for optimal performance. Eight common FGFR3 mutations in 3 exons have been associated with bladder cancer. Analysis of mutational status for each single mutation required 8 amplification steps, which were costly and time consuming. We have now developed a real-time multiplexed FGFR3 assay, generating a cost-effective, clinically applicable assay for the detection of FGFR3 mutations in urine. Methods: Our approach involves a two-step PCR amplification process. The initial round generates exon specific PCR products, which are then used as template for real-time PCR mutation detection utilizing locked nucleic acid (LNA) oligonucleotides. The LNA suppress wild-type DNA amplification. To convert our existing FGFR3 assay to a multiplex format, primary amplifications of exons 7, 10, and 15 were combined into a single real-time PCR assay for exon specific amplification and DNA quantitation. The LNA-mediated mutation detection was then converted from 4 reactions to 2 duplex amplifications. All multiplex assays were carried out on the Roche LC 480 real-time PCR platform. Results: To validate the new multiplex format, FGFR3 multiplex analysis was performed on DNA isolated from 50 Ta stage bladder tumors. FGFR3 mutations were detected in 90% (48/50) of the tumors. To directly compare performance with single mutation analysis, 40 urine samples previously analyzed using the singleplex format were again tested using the multiplex FGFR3 assay. 100% concordance was seen between the two assay formats. Conclusions: By multiplexing the FGFR3 mutation analysis we reduced the number of amplification steps, improving assay turnaround time and throughput, without compromising assay performance. The FGFR3 multiplex analysis provides a robust, cost-effective DNA assay that in combination with MMP protein analysis delivers a clinically applicable assay with optimal performance. [Table: see text]

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.

The importance of β globin deletion analysis in the evaluation of patients with β thalassemia

Beta globin deletion/duplication analysis may serve as a useful adjunct to sequence analysis. Our purpose was to develop a robust assay for beta globin deletion/duplication analysis and determine its role in evaluating patients with beta thalassemia. A single tube semi-quantitative fluorescent PCR assay capable of detecting deletions and duplications in the beta globin cluster and the associated locus control region (LCR) was developed and validated. Six hundred seventy one de-identified samples submitted for beta globin sequence analysis were tested for deletions and duplications of the beta globin cluster. Twenty-two deletions were detected (3%, 22/671). Seventeen of the 22 (82%) deletion samples were negative for mutations in the whole gene sequencing assay. For 5 of the samples, homozygous point mutations were inferred by beta globin sequencing. Among the deletions detected, 11 (50%) involved only the beta globin gene (5 covering the entire gene, 2 spanning the 5' end of the gene and 4 encompassing the 3' end of the gene). Ten samples (45%) were heterozygous delta-beta deletions spanning both the delta globin and beta globin genes. One patient with a single deletion had Hb Lepore. Beta globin deletion/duplication analysis is necessary to correctly identify the genotype in some patients being evaluated for beta thalassemia.

Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)(-1), whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.