scholarly journals Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus

2019 ◽  
Vol 269 ◽  
pp. 13-17
Author(s):  
Leyi Wang ◽  
Therese E. Eggett ◽  
Saraswathi Lanka ◽  
Richard L. Fredrickson ◽  
Ganwu Li ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Encephalomyelitis Virus ◽  
Porcine Hemagglutinating Encephalomyelitis Virus

scholarly journals Establishment of a Real-time RT-PCR Assay for Avian Encephalomyelitis Virus

2016 ◽  
Vol 72 (7) ◽  
pp. 418-422
Author(s):  
Qinghong Xue ◽  
Hui Guo ◽  
Zhongze Feng ◽  
Jia Wang
Keyword(s):  
Real Time ◽  
Disease Virus ◽  
Rapid Detection ◽  
Prevention And Control ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Agar Diffusion Test ◽  
Encephalomyelitis Virus ◽  
And Control

Avian encephalomyelitis is an epidemic disease caused by avian encephalomyelitis virus (AEV), which exerts egg drop, slow growth, decreased egg hatchability, rapid tremors and ataxia of the head and neck in chicks and laying hens. The availability of a robust diagnostic assay to confirm the infection of AEV is important for its prevention and control. Thus, this study aimed to develop and validate a rapid Real-time RT-PCR assay for the detection and quantification of AEV. The authors developed and validated a TaqMan-based fluorescent real-time RT-PCR with sensitivity, specificity and reproducibility for rapid detection of EAV. This assay was then applied on 52 clinical samples from 37 suspected chickens. The minimal detection limit of this method was 20 copies. No cross-reactivity with Newcastle disease virus (NDV), avian influenza virus (AIV), Marek’s disease virus (MDV) or avian leukosis virus (ALV) was observed. The intra- and inter-assay coefficients of variation were both below 2%. Of the 52 clinical samples analyzed using this assay, 33 samples were positive for AEV, yielding an agreement rate of 91.2% with RT-PCR and 88.2% with the agar diffusion test. In conclusion, we have successfully developed and validated a sensitive Real-time RT-PCR for rapid detection of AEV, which could be used in the early diagnosis, prevention and control, and epidemiological investigation of AEV.

Development of a SYBR Green real-time RT-PCR assay for the detection of avian encephalomyelitis virus

2014 ◽  
Vol 206 ◽  
pp. 46-50 ◽  
Author(s):  
Qingtian Liu ◽  
Zengqi Yang ◽  
Huafang Hao ◽  
Shenli Cheng ◽  
Wentao Fan ◽  
...  
Keyword(s):  
Real Time ◽  
Sybr Green ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Encephalomyelitis Virus

scholarly journals Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

2021 ◽  
pp. 104063872199481
Author(s):  
Yixin Xiao ◽  
Fan Yang ◽  
Fumin Liu ◽  
Hangping Yao ◽  
Nanping Wu ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Influenza A ◽  
Minor Groove Binder ◽  
Rt Pcr ◽  
Influenza A Viruses ◽  
Pcr Assay ◽  
Infectious Dose ◽  
The Matrix ◽  
Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

2013 ◽  
Vol 189 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Yong Yan ◽  
Heng-hui Wang ◽  
Lei Gao ◽  
Ji-mei Ji ◽  
Zhi-jie Ge ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Human Norovirus ◽  
One Step

Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

2010 ◽  
Vol 47 (1) ◽  
pp. 54-59 ◽  
Author(s):  
Sylvie Pillet ◽  
Geneviève Billaud ◽  
Shabir Omar ◽  
Bruno Lina ◽  
Bruno Pozzetto ◽  
...  
Keyword(s):  
Real Time ◽  
R Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Clinical Specimens

scholarly journals Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 641-644 ◽  
Author(s):  
Manphool S. Fageria ◽  
Mathuresh Singh ◽  
Upeksha Nanayakkara ◽  
Yvan Pelletier ◽  
Xianzhou Nie ◽  
...  
Keyword(s):  
Real Time ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Current Season ◽  
Seed Market ◽  
Polymerase Chain ◽  
Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S134-S135
Author(s):  
T Berent ◽  
T Rothstein ◽  
S Buckwalter ◽  
R Patel
Keyword(s):  
Real Time ◽  
Whole Blood ◽  
New Technologies ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Bartonella Species ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Hybridization Probes ◽  
Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

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