scholarly journals A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation ofSugarcane Streak Mosaic Virus

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Wei-Lin Fu ◽  
Sheng-Ren Sun ◽  
Hua-Ying Fu ◽  
Ru-Kai Chen ◽  
Jin-Wei Su ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Taqman Probe ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Total Rna ◽  
Qrt Pcr ◽  
Efficient Detection ◽  
One Step

Sugarcane mosaic disease is caused by theSugarcane streak mosaic virus(SCSMV; genusPoacevirus, familyPotyviridae) which is common in some Asian countries. Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane. Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences. Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmidin vitroand total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR. We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2 pg of total RNA with good reproducibility. The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR. A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms. SCSMV-free samples were verified by infection withSugarcane mosaic virus(SCMV) orSorghum mosaic virus(SrMV) or a combination of both. The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV.

Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings

2014 ◽  
Vol 58 (01) ◽  
pp. 81-85 ◽  
Author(s):  
A. ZARZYŃSKA ◽  
M. JEŻEWSKA ◽  
K. TRZMIEL ◽  
B. HASIÓW-JAROSZEWSKA
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Barley Stripe Mosaic Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step ◽  
Virus Strains

scholarly journals A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV)

2013 ◽  
Vol 10 (1) ◽  
pp. 173 ◽  
Author(s):  
Wenwen Liu ◽  
Xiaojuan Zhao ◽  
Peng Zhang ◽  
Thi Mar ◽  
Yan Liu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Yellow Mosaic Virus ◽  
Wheat Yellow Mosaic Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

scholarly journals Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil

Sensors ◽  
2012 ◽  
Vol 12 (12) ◽  
pp. 16685-16694 ◽  
Author(s):  
Jin-Guang Yang ◽  
Feng-Long Wang ◽  
De-Xin Chen ◽  
Li-Li Shen ◽  
Yu-Mei Qian ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Real Time ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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

scholarly journals Real-Time Reverse Transcription-Polymerase Chain Reaction Detection of Newcastle Disease Virus Using Light upon Extension Fluorogenic Primers

2007 ◽  
Vol 19 (4) ◽  
pp. 400-404 ◽  
Author(s):  
Márta Antal ◽  
Tibor Farkas ◽  
Péter Germán ◽  
Sándor Belák ◽  
István Kiss
Keyword(s):  
Newcastle Disease Virus ◽  
Real Time ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Plasmid Copy Number ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Plasmid Copy ◽  
Infectious Dose ◽  
Efficient Detection

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10+1.2 egg infectious dose (EID)50/0.2 ml and 10+2.2 EID50/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.

Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay

2017 ◽  
Vol 33 ◽  
pp. 8-10 ◽  
Author(s):  
Jingfang Chen ◽  
Rusheng Zhang ◽  
Xinhua Ou ◽  
Dong Yao ◽  
Zheng Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Rapid Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step
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