scholarly journals Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp

2003 ◽  
Vol 69 (11) ◽  
pp. 6723-6730 ◽  
Author(s):  
Rainer Kurmayer ◽  
Thomas Kutzenberger
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Total Population ◽  
Intergenic Spacer ◽  
Threshold Level ◽  
Logarithmic Scale ◽  
Cycle Number ◽  
Intergenic Spacer Region ◽  
Cell Numbers ◽  
The Mean

ABSTRACT The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.

scholarly journals Characterization of a novel Mycoplasma cynos real-time PCR assay

2019 ◽  
Vol 32 (6) ◽  
pp. 793-801 ◽  
Author(s):  
Rebecca L. Tallmadge ◽  
Renee Anderson ◽  
Patrick K. Mitchell ◽  
Zachary C. Forbes ◽  
Brenda Werner ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intergenic Spacer ◽  
High Specificity ◽  
Mycoplasma Species ◽  
Limit Of Quantification ◽  
Clinical Specimens ◽  
Intergenic Spacer Region ◽  
Tuf Gene ◽  
Gene Assay

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3–97.9% ( r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1–122.5% ( r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

New PCR-based assay for the identification of Pectobacterium carotovorum causing potato soft rot

Plant Disease ◽  
2021 ◽  
Author(s):  
M. Belén Suárez ◽  
Marta Diego ◽  
F. J. Feria ◽  
M J Martín-Robles ◽  
Sergio Moreno ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Intergenic Spacer ◽  
Soft Rot ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Intergenic Spacer Region

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management, since different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is P. carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region (ITS) of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of P. versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen P. parmentieri. This should be useful for the routine diagnosis of potato soft rot.

Conventional and real-time PCR assays for specific detection and quantification of Fusarium oxysporum f. sp. ciceris in plants using intergenic spacer region-based marker

Biologia ◽  
2015 ◽  
Vol 70 (3) ◽  
Author(s):  
Kumari Priyanka ◽  
Sunil C. Dubey ◽  
Arun K. Singh
Keyword(s):  
Fusarium Oxysporum ◽  
Host Plant ◽  
Real Time ◽  
Real Time Pcr ◽  
Infected Plant ◽  
Intergenic Spacer ◽  
Infected Host ◽  
Pcr Assay ◽  
Intergenic Spacer Region ◽  
Plant Samples

AbstractFusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris (Foc) is one of the most important fungal diseases worldwide. The detection of the pathogen at reasonable time period is of great importance, which requires rapid and sensitive detection methods. The intraspecific divergence sequences found in the intergenic spacer region (IGS) were selected and utilized with the aim to develop a molecular marker specifically to identify the Foc. A marker set, ISR52 F1 and R1 developed, was tested for their specificity as well as sensitivity using conventional as well as real-time polymerase chain reaction (PCR). The specificity of the marker was tested against Foc, other Fusarium species which are closely related to Foc as well as with artificially infected host plant samples. The detection limits of conventional PCR assay was up to 100 pg of infected plant DNA. It proved possible to amplify the IGS region in different portion of a Foc infected host plant by this PCR method. Furthermore, the real-time assay showed more sensitivity and was able to detect the pathogen in infected chickpea plant samples at the DNA concentration of 5 pg. A single melting peak obtained at 87.5°C showed the specificity of the marker towards Foc. Thus, real-time PCR assay proved their potentiality for same-day diagnosis of fungal infection and can be used as a rapid and effective procedure for routine detection and identification of Foc in chickpea samples.

scholarly journals Prevalence and phylogenetic analysis of Mycoplasma synoviae strains isolated from Polish chicken layer flocks

2019 ◽  
Vol 63 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Olimpia Kursa ◽  
Grzegorz Tomczyk ◽  
Anna Sawicka
Keyword(s):  
Phylogenetic Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Material ◽  
Clinical Signs ◽  
Intergenic Spacer ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Mycoplasma Synoviae ◽  
Intergenic Spacer Region

AbstractIntroduction:Mycoplasma synoviae(MS) is a chicken pathogen of major economic importance.Material and Methods:Between 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S–23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect thevlhAgene.Results:MS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of PolishM. synoviaestrains using a partial sequence of thevlhAgene showed nine genotypes (A–I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2).Conclusion:These data showed the country’s isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data onM. synoviaeinfection in layer chickens in Poland.

Development of a real-time PCR for detection of the oyster pathogen Nocardia crassostreae based on its homogeneous 16S–23S rRNA intergenic spacer region

2013 ◽  
Vol 114 (2) ◽  
pp. 120-127 ◽  
Author(s):  
Noèlia Carrasco ◽  
Ineke Roozenburg ◽  
Michal Voorbergen-Laarman ◽  
Naoki Itoh ◽  
Marc Y. Engelsma
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Intergenic Spacer Region

scholarly journals ORAL HIV-ASSOCIATED KAPOSI SARCOMA: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY AND qPCR TECHNIQUES FOR DETECTION OF HHV8

2015 ◽  
Vol 21 (1) ◽  
pp. 29
Author(s):  
Priscila Lie Tobouti ◽  
Juliana Seo ◽  
Michella Bezerra Lima ◽  
Bruno Tavares Sedassari ◽  
Norberto Nobuo Sugaya ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Kaposi Sarcoma ◽  
Positive Staining ◽  
Extraction Protocol ◽  
Simple Phenol ◽  
Two Samples ◽  
Hematoxylin And Eosin ◽  
The Mean

<p><strong>Objective: </strong>To compare the diagnostic accuracy of immunohistochemistry compared to real-time PCR (using a simple phenol-chloroform DNA extraction protocol) in the detection of HHV8 in small oral biopsies of Kaposi sarcoma. Also to validate the use of this DNA extraction protocol to extract HHV8 DNA.</p><p><strong>Material and methods:</strong> Seventeen cases of oral KS were examined. Data including gender, age, and anatomic location were obtained from patient´s records and histological sections stained with hematoxylin and eosin (H&amp;E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.</p><p><strong>Results: </strong>All the patients were HIV positive, the mean age was 35.5 years old, and the affected oral sites were palate (47%), gingiva (29.4%), tongue (11.8%), lip (5.9%), and oral floor (5.9%). Fifteen samples showed positive staining for HHV8 and only two samples were negative. The same results were observed using real-time PCR HHV8-DNA detection.</p><p><strong>Relevance: </strong>Our findings suggest that immunohistochemistry is faster and cheaper to perform than real-time PCR and was shown to have similar levels of sensitivity and accuracy for detection of HHV8 even in small biopsies. Additionally DNA extraction using a non-commercial kit, as done in this study can further reduce the costs to a pathology service.</p>

Clonal diversity in overwintering populations of Schizaphis graminum (Homoptera: Aphididae)

1994 ◽  
Vol 84 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Kevin A. Shufran ◽  
Gerald E. Wilde
Keyword(s):  
Total Population ◽  
Intergenic Spacer ◽  
Clonal Diversity ◽  
Population Diversity ◽  
Schizaphis Graminum ◽  
Molecular Fingerprinting ◽  
Intergenic Spacer Region ◽  
The Usa ◽  
High Degree ◽  
Total Diversity

AbstractThe intergenic spacer region of the rRNA cistron was used as a molecular fingerprinting probe to study clonal diversity in Schizaphis graminum (Rondani). A high degree of clonal diversity was found in overwintering populations on wheat from Kansas, Texas and Oklahoma. Out of 184 individuals tested, 132 unique maternal lineages wereidentified. Overall, clonal diversity of overwintering populations was no less than in spring and summer populations. However, in some fields there were more instances where individuals of the same genotype were identified. By partitioning the total population diversity into various sampling components (fields, counties and states), 93.1% of the total diversity was found among individual S. graminum within fields. Overwintering of many genetically distinct clones as parthenogenetic morphs is one mechanism by which clonal diversity is maintained in populations of S. graminum. The incidence of clonal diversity further substantiates the importance of the S. graminum holocycle for generating genetic heterogeneity in the USA.

scholarly journals Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 1941-1947 ◽  
Author(s):  
Alexander J. Ryncarz ◽  
James Goddard ◽  
Anna Wald ◽  
Meei-Li Huang ◽  
Bernard Roizman ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Genital Tract ◽  
Clinical Samples ◽  
Pcr Assay ◽  
The Mean ◽  
Simplex Virus

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

scholarly journals Restriction Site–Specific Methylation Studies of Imprinted Genes with Quantitative Real-Time PCR

2008 ◽  
Vol 54 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Sara Bruce ◽  
Katariina Hannula-Jouppi ◽  
Cecilia M Lindgren ◽  
Marita Lipsanen-Nyman ◽  
Juha Kere
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Uniparental Disomy ◽  
Restriction Enzymes ◽  
Chromosome 7 ◽  
Imprinted Genes ◽  
Quantitative Real Time Pcr ◽  
Large Patient ◽  
The Mean

Abstract Background: Epigenetic studies, such as the measurement of DNA methylation, are important in the investigation of syndromes influenced by imprinted genes. Quick and accurate quantification of methylation at such genes can be of appreciable diagnostic aid. Methods: We first digested genomic DNA with methylation-sensitive restriction enzymes and used DNA without digestion as a control and nonmethylated λ DNA as an internal control for digestion efficiency. We then performed quantitative real-time PCR analyses with 6 unique PCR assays to investigate 4 imprinting control regions on chromosomes 7 and 11 in individuals with uniparental disomy of chromosome 7 (UPD7) and in control individuals. Results: Our validation of the method demonstrated both quantitative recovery and low methodologic imprecision. The imprinted loci on chromosome 7 behaved as expected in maternal UPD7 (100% methylation) and paternal UPD7 (&lt;10% methylation). In controls, the mean (SD) for percent methylation at 2 previously well-studied restriction sites were 46% (6%) for both H19 and KCNQ1OT1, a result consistent with the previously observed methylation rate of approximately 50%. The methylation percentages of all investigated imprinted loci were normally distributed, implying that the mean and SD can be used as a reference for screening methylation loss or gain. Conclusion: The investigated loci are of particular importance for investigating the congenital Silver–Russell and Beckwith–Wiedemann syndromes; however, the method can also be applied to other imprinted regions. This method is easy to set up, has no PCR bias, requires small amounts of DNA, and can easily be applied to large patient populations for screening the loss or gain of methylation.

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