Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Canine Enteric Bacterial Pathogens

2015 ◽  
Vol 32 (2) ◽  
pp. 154
Author(s):  
Hye-Jin Jang ◽  
Jae-Ik Han ◽  
Hyo-Min Kang ◽  
Ki-Jeong Na
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Bacterial Pathogens ◽  
Simultaneous Detection ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Quantification of Venturia inaequalis Growth in Malus × domestica with Quantitative Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1791-1797 ◽  
Author(s):  
Michele Gusberti ◽  
Andrea Patocchi ◽  
Cesare Gessler ◽  
Giovanni A. L. Broggini
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Malus Domestica ◽  
Venturia Inaequalis ◽  
Simultaneous Detection ◽  
Scab Resistance ◽  
Infected Leaf ◽  
Chain Reaction ◽  
Limit Of Quantification ◽  
Polymerase Chain

A quantitative real-time polymerase chain reaction (qPCR) was developed and validated for quantification of Venturia inaequalis in infected leaf tissue of Malus × domestica. The method is based on dual-labeled hybridization probes, allowing simultaneous detection of host and pathogen DNA within one single reaction. Limit of quantification for the pathogen was 0.5 pg per reaction and, for the host, reached 5 pg per reaction. The fungal growth measured in four apple cultivars 2 weeks after inoculation significantly correlated with their different level of scab resistance and allowed the observation of ontogenic resistance. After sporulation on the youngest leaf, fungal biomass in susceptible ‘Gala’ was 118 times higher than in resistant ‘Florina’ and ‘Discovery’ while intermediate values were found with the intermediate susceptible ‘Milwa’. Correlation was also observed between severity classes obtained by visual scoring of symptoms and qPCR results. Moreover, qPCR demonstrated validity of the developed method as a disease severity forecast tool 10 days after the pathogen's inoculation and prior to the appearance of the symptoms. Applications of the methodology can include the quantification of scab resistance during breeding programs, evaluation of fungicide and biocontrol efficacy, and quantification of the fitness of different pathogenic strains.

A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

2021 ◽  
Author(s):  
Yang Pan ◽  
Jing Chen ◽  
Junhuang Wu ◽  
Yongxia Wang ◽  
Junwei Zou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rapid Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Canine Kobuvirus

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

scholarly journals Influenza species and subtypes circulation among hospitalized patients in Laleh hospital during two influenza seasonal (2016-2017 and 2017-2018) using a multiplex Real Time-Polymerase Chain Reaction

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Iman Rezaee Azhar ◽  
Minoo Mohraz ◽  
Masoud Mardani ◽  
Mohammad Ali Tavakoli ◽  
Amin Ehteshami Afshar ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Hospitalized Patients ◽  
P Value ◽  
Chain Reaction ◽  
Short Period ◽  
The Mean ◽  
Polymerase Chain

The introduction of polymerase chain reaction (PCR) techniques has improved the detection of respiratory viruses, particularly with the use of multiplex real-time technique with the capability of simultaneous detection of various pathogens in a single reaction. The aim of this study was to apply the above technology for the diagnosis of influenza infections and at the same time to differentiate between common flu species between hospitalized patients in Laleh hospital (Iran) between two flu seasons (2016- 2017 and 2017-2018). Different respiratory specimens were collected from 540 patients from a period of December 2016 to May 2018 and were sent to the laboratory for molecular diagnosis. RNAs were extracted and subsequently, a multiplex real time PCR identifying flu A, flu B and typing flu A (H1N1) was carried out. The mean age of patients was 47.54±23.96. 216 (40%) and 321 (60%) of subjects were male and female, respectively. 219 out of 540 (40.5%) were positive for influenza infection including flu A (n=97, 44.3%), flu A (H1N1) (n=45, 20.7%) and flu B (n=77, 35%). Flu A was the dominant species on 2016-2017 and flu B was the major species on 2017-2018. Flu A (H1N1) was comparable in both time periods. Flu infections were most frequently diagnosed in age groups 21-40. Flu-positive patients suffered more from body pain and sore throat than flunegative patients with significant statistical difference (P values <0.001). The mean duration of hospitalization was shorter for flu-positive patients (P value = 0.016). Application of multiplex real time PCR could facilitate the influenza diagnosis in a short period of time, benefiting patients from exclusion of bacterial infections and avoiding unnecessary antibiotic therapy. Influenza diagnosis was not achieved in up to 60% of flu-like respiratory infections, suggesting the potential benefit of adopting the same methodology for assessing the involvement of other viral or/and bacterial pathogens in those patients.

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