Development and application of a reverse transcriptase droplet digital PCR (RT-ddPCR) for sensitive and rapid detection of Japanese encephalitis virus

Wu, Xulong; Lin, Hua; Chen, Shijie; Lu, Xiao; Yang, Miao; An, Wei; Wang, Yin; Yao, Xueping; Yang, Zexiao

doi:10.1016/j.jviromet.2017.06.015

Cited by 32 publications

(18 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3b). The viral RNA copies of both strains extracted from the suspensions (culture supernatants and infected BSR T7/5 cells) 36 h post infection at an MOI of 0.1 were measured in triplicate by qRT-PCR [22]. There were an average of 18 copies/TCID 50 in the parent strain, and an average of 20 copies/TCID 50 in the rescued virus.…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2

Jin

Nie³

et al. 2019

Virus Genes

View full text Add to dashboard Cite

Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3′ UTR cDNA of JEV for authentic 3′ UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.

show abstract

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2

Jin

Nie³

et al. 2019

Virus Genes

View full text Add to dashboard Cite

show abstract

“…52 Digital PCR is a novel PCR technology, which so far has been used only for porcine pathogen detection in a few published studies. 51,54 It is a highly accurate and sensitive method that enables absolute quantification without the need of a standard curve. However, the dPCR machines and reagents are still expensive, hence, this technology is not a preferred tool in diagnostic laboratories at the moment.…”

Section: Research-article2019mentioning

confidence: 99%

Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

et al. 2019

View full text Add to dashboard Cite

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

show abstract

“…Digital PCR overcomes the need for a standard curve, and it is increasingly used for DNA/RNA viral quantification, in human and animal health (11)(12)(13)(14)(15)(16)(17). Bluetongue (BT) is an Office International des Epizooties (OIE)-listed infectious disease of domestic and wild ruminants (18), transmitted mainly through the bites of Culicoides midges.…”

Section: Introductionmentioning

confidence: 99%

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples

et al. 2020

View full text Add to dashboard Cite

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (10 5.43 TCID 50 /ml up to 10 −0.57 TCID 50 /ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R 2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10 −0.67 TCID 50 /ml (0.72 copies/µl) and 10 0.03 TCID 50 /ml (3.05 copies/µl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.

show abstract

Development and application of a reverse transcriptase droplet digital PCR (RT-ddPCR) for sensitive and rapid detection of Japanese encephalitis virus

Cited by 32 publications

References 18 publications

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2

Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2

Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples

Contact Info

Product

Resources

About