scholarly journals Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

2011 ◽  
Vol 77 (18) ◽  
pp. 6323-6330 ◽  
Author(s):  
Steen Nordentoft ◽  
Susanne Kabell ◽  
Karl Pedersen
Keyword(s):  
Real Time ◽  
Cost Effective ◽  
Sybr Green ◽  
Detection Methods ◽  
Intracellular Pathogens ◽  
Microscopical Examination ◽  
Screening Assay ◽  
Content Type ◽  
Specific Pcr ◽  
Pcr Assays

ABSTRACTInfections caused by members of theChlamydiaceaefamily have long been underestimated due to the requirement of special laboratory facilities for the detection of this group of intracellular pathogens. Furthermore, new studies of this group of intracellular pathogens have revealed that host specificity of different species is not as clear as recently believed. As most members of the genusChlamydophilahave shown to be transmissible from animals to humans, sensitive and fast detection methods are required. In this study, SYBR green-based real-time assays were developed that detect all members ofChlamydiaceaeand differentiate the most prevalent veterinaryChlamydophilaspecies:Cp. psittaci,Cp. abortus,Cp. felis, andCp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs.Cp. psittaciwas detected in 47 birds,Cp. feliswas found in 10 cats,Cp. caviaewas found in one guinea pig, andCp. abortuswas detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation with a highly sensitive family-specific PCR, we were able to screen forChlamydiaceaein veterinary specimens and confirm the species in positive samples with additional PCR assays.

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.

scholarly journals Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

2012 ◽  
Vol 78 (10) ◽  
pp. 3630-3637 ◽  
Author(s):  
Karol Krak ◽  
Martina Janoušková ◽  
Petra Caklová ◽  
Miroslav Vosátka ◽  
Helena Štorchová
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Glomus Intraradices ◽  
Large Subunit ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Content Type ◽  
Copy Numbers ◽  
Pcr Assays

ABSTRACTReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates ofGlomus intraradicessensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

Specific PCR and real-time PCR assays for detection and quantitation of ‘Candidatus Phytoplasma phoenicium’

2015 ◽  
Vol 29 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Maan Jawhari ◽  
Peter Abrahamian ◽  
Ali Abdel Sater ◽  
Hana Sobh ◽  
Patil Tawidian ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Specific Pcr ◽  
Pcr Assays

scholarly journals Real-time structural health monitoring for concrete beams: a cost-effective ‘Industry 4.0’ solution using piezo sensors

2020 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Arka Ghosh ◽  
David John Edwards ◽  
M. Reza Hosseini ◽  
Riyadh Al-Ameri ◽  
Jemal Abawajy ◽  
...  
Keyword(s):  
Structural Health Monitoring ◽  
Real Time ◽  
Health Monitoring ◽  
Industry 4.0 ◽  
Concrete Beams ◽  
Low Cost ◽  
Cost Effective ◽  
Content Type ◽  
Structural Health ◽  
Non Destructive

PurposeThis research paper adopts the fundamental tenets of advanced technologies in industry 4.0 to monitor the structural health of concrete beam members using cost-effective non-destructive technologies. In so doing, the work illustrates how a coalescence of low-cost digital technologies can seamlessly integrate to solve practical construction problems.Design/methodology/approachA mixed philosophies epistemological design is adopted to implement the empirical quantitative analysis of “real-time” data collected via sensor-based technologies streamed through a Raspberry Pi and uploaded onto a cloud-based system. Data was analysed using a hybrid approach that combined both vibration-characteristic-based method and linear variable differential transducers (LVDT).FindingsThe research utilises a novel digital research approach for accurately detecting and recording the localisation of structural cracks in concrete beams. This non-destructive low-cost approach was shown to perform with a high degree of accuracy and precision, as verified by the LVDT measurements. This research is testament to the fact that as technological advancements progress at an exponential rate, the cost of implementation continues to reduce to produce higher-accuracy “mass-market” solutions for industry practitioners.Originality/valueAccurate structural health monitoring of concrete structures necessitates expensive equipment, complex signal processing and skilled operator. The concrete industry is in dire need of a simple but reliable technique that can reduce the testing time, cost and complexity of maintenance of structures. This was the first experiment of its kind that seeks to develop an unconventional approach to solve the maintenance problem associated with concrete structures. This study merges industry 4.0 digital technologies with a novel low-cost and automated hybrid analysis for real-time structural health monitoring of concrete beams by fusing several multidisciplinary approaches into one integral technological configuration.

scholarly journals Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of the DEFB1 Gene with Early-Onset Periodontal Disease

2004 ◽  
Vol 11 (4) ◽  
pp. 766-769 ◽  
Author(s):  
Michele Boniotto ◽  
Manzour Hernando Hazbón ◽  
William James Jordan ◽  
Greig Patrick Lennon ◽  
Joyce Eskdale ◽  
...  
Keyword(s):  
Periodontal Disease ◽  
Real Time ◽  
Early Onset ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Similar Distribution ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Specific Allele

ABSTRACT A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The −44 C→G transversion in the DEFB1 gene (which encodes human β-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the −44 SNP. We used an HP assay to study the distribution of the −44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the −44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.

scholarly journals Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris

2017 ◽  
Vol 55 (8) ◽  
pp. 2445-2452 ◽  
Author(s):  
Milena Kordalewska ◽  
Yanan Zhao ◽  
Shawn R. Lockhart ◽  
Anuradha Chowdhary ◽  
Indira Berrio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Related Species ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Invasive Infections ◽  
Candida Lusitaniae ◽  
Pcr Assays ◽  
Phenotypic Tests

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae . Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.

scholarly journals Novel Dual Labeled Fluorescence Probe Based Assay to Measure the Telomere Length

2018 ◽  
Author(s):  
Itty Sethi ◽  
Gh. Rasool Bhat ◽  
Rakesh Kumar ◽  
Ekta Rai ◽  
Swarkar Sharma
Keyword(s):  
Polymerase Chain Reaction ◽  
Telomere Length ◽  
Fluorescence Probe ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Sybr Green ◽  
Telomeric Dna ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Polymerase Chain

ABSTRACTTelomeres are highly repetitive regions capping the chromosomes and composed of multiple units of hexa-nucleotides, TTAGGG, making their quantification difficult. Most of the methods developed to estimate telomeres are extensively cumbersome and expensive. The quantitative polymerase chain reaction (qPCR) based assay is relatively easy and cheaper method that applies SyBr Green dye chemistry to measure telomere length. As SyBr Green dye fluoresces after intercalation into the dsDNA, lack of differentiation between specific PCR target products and unspecific products is a limitation and it affects accuracy in quantitation of telomeres. To overcome the limitations of SyBr Green, we developed a dual labeled fluorescence probe based quantitative polymerase chain reaction (qPCR) to measure the telomere length. This robust, accurate and highly reproducible (R2=0.96) proprietary method (patent pending), yet cost effective and easy, utilizes a probe that targets specifically the telomeric DNA.

scholarly journals Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

2016 ◽  
Vol 55 (2) ◽  
pp. 526-534 ◽  
Author(s):  
Ciro Martins Gomes ◽  
Mariana Vicente Cesetti ◽  
Natália Aparecida de Paula ◽  
Sebastián Vernal ◽  
Gaurav Gupta ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Conventional Pcr ◽  
Content Type ◽  
Tegumentary Leishmaniasis ◽  
Precise Diagnosis ◽  
American Tegumentary Leishmaniasis ◽  
Pcr Techniques ◽  
Composite Reference Standard

ABSTRACTThe precise diagnosis of American tegumentary leishmaniasis (ATL) is an essential task due to the disease's associated morbidity. A noninvasive, extremely sensitive, and highly specific exam is critical, particularly for mucosal leishmaniasis (ML), in which a low parasite quantity is expected. We aimed to compare the diagnostic accuracy of swab and biopsy sample analysis using SYBR Green- and TaqMan-based real-time PCR (qPCR) assays with that of a composite reference standard consisting of the Montenegro skin test, serology, histopathology, smears, culture, and conventional PCR. In total, 55 patients with ATL (ML, 18 patients; cutaneous leishmaniasis [CL], 37 patients) and 36 patients without ATL were studied. qPCR analysis of swabs was more accurate when using SYBR Green (87.88%; 95% confidence interval [CI], 77.86 to 93.73 patients) than when using TaqMan (78.79%; 95% CI, 67.49 to 86.92%) (P= 0.031). SYBR Green (84.72%; 95% CI, 74.68 to 91.25%) was also more accurate than TaqMan (73.61%; 95% CI, 62.42 to 82.41%) for biopsy samples (P= 0.008). All qPCR methods were 100% specific. Swabs and biopsy specimens had similar sensitivity when using the same chemistry (P= 0.125 for SYBR Green andP= 0.625 for TaqMan). Moreover, qPCR achieved better performance than most existing techniques used for the diagnosis of ATL and also detected theLeishmaniaparasite in a greater proportion of patients than the associated histopathology, smear, culture, and conventional PCR techniques did. Swabs therefore represent a useful diagnostic tool because they not only are noninvasive but also can achieve an accuracy similar to that of biopsy samples. The high accuracy of SYBR Green-based qPCR may also reduce the requirement for associated parasitological tests for ATL diagnosis.

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