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.

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.

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.

scholarly journals An Event-Specific Real-Time PCR Method for Measuring Transgenic Lysozyme Goat Content in Trace Samples

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 925
Author(s):  
Wenting Xu ◽  
Jinjie Cui ◽  
Biao Liu ◽  
Litao Yang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Goat Milk ◽  
High Specificity ◽  
Limit Of Quantification ◽  
Transgenic Goat ◽  
Potential Applicability ◽  
Pcr Method

Lysozymes are used in sterilisation, antisepsis, dairy additives, inflammation, and cancer. One transgenic goat line expressing high levels of human lysozyme (hLZ) in goat milk has been developed in China. Herein, we established an event-specific real-time polymerase chain reaction (real-time PCR) method to detect the transgenic hLZ goat line. The developed method has high specificity, sensitivity and accuracy, and a wide quantitative dynamic range. The limit of detection and limit of quantification was 5 and 10 copies per reaction, respectively. The practical sample analysis results showed that the method could identify and quantify transgenic lysozyme content in trace samples in routine lab analyses. Furthermore, the potential applicability in risk assessment, such as molecular characterisation and gene horizontal transfer, was confirmed. We believe that this method is suitable for the detection of transgenic hLZ goat line and its derivate.

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

Identification of L. casei and L. rhamnosus in the dairy products using Real-Time PCR assay

2015 ◽  
Vol 4 (5) ◽  
pp. 222-225
Author(s):  
K. G. Li ◽  
G. P. Pogossian ◽  
A. K. Moldagulova ◽  
E. E. Bekenova ◽  
A. Abdikadirova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dairy Products ◽  
Production Control ◽  
High Efficiency ◽  
High Specificity ◽  
Industrial Test ◽  
Fermented Dairy Products ◽  
Species Specific ◽  
Fermented Dairy

  Lactobacilli are essential and important biological objects used in food pro-duction and medicine. One of the sufficient problems is fast, reliable and highly specific identification of lactobacilli in the scientific research and cur-rent production control. We represent two species-specific real-time PCR in the present study to discriminate L. rhamnosus and L. casei basing on the unique peptidoglycan-hydrolase genes p40 and p75 respectively. PCR pri-mers and probes were designed to provide high specificity discrimination via high temperature of PCR annealing stage. High efficiency of the reactions is provided by the size of amplified DNA fragments minimization. Reliable re-producibility of the target sequences amplification and fluorescence detec-tion provide a basis for the future creation of industrial test-systems for op-erational control in the production of fermented dairy products.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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