Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

Jia, Peng; Purcell, Maureen K.; Pan, Guang; Wang, Jinjin; Kan, Shihai; Yin, Lifang; Zheng, Xuelian; Shi, Xiujie; He, Jianping; Yu, Li; Qun-yi, Hua; Lu, Tikang; Lan, Wensheng; Winton, James R.; Jin, Ningyi; Liu, Hong

doi:10.1016/j.jviromet.2017.03.010

Cited by 17 publications

(17 citation statements)

References 37 publications

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“…RNA targets additionally have to be converted into cDNA prior to PCR analysis . Alternatively, DNAs or RNAs of interest can be detected directly by using a homogeneous or solid‐phase fluorescence assay . Thanks to the advantageous properties of the new NP probes described above and the simplicity of the amplification‐free hybridization assay, it is more time‐ and cost‐effective than regular PCR settings, given that the target has a concentration above the detection limit for the particular assay.…”

Section: Methodsmentioning

confidence: 99%

“…Thanks to the advantageous properties of the new NP probes described above and the simplicity of the amplification‐free hybridization assay, it is more time‐ and cost‐effective than regular PCR settings, given that the target has a concentration above the detection limit for the particular assay. This is the case in, for example, cell line DNA analysis and pathogen‐specific DNA/RNA detection in biofluids …”

Section: Methodsmentioning

confidence: 99%

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New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

et al. 2017

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For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification-free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra-bright macrofluorophores of 9-84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target-specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

et al. 2017

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“…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%

“…Maan et al (32) transcribed from recombinant pGEMT plasmid RNA BT using the mMessage mMachine T7 Ultra Kit (Life Technologies). Overall, the use of different calibration standards in different performing assays can lead to nonreproducible results between laboratories, even when testing the same material (12,33).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…Both methods have benefits, but the droplet digital PCR obtains a greater number of partitions that provide a more reliable quantification (Basu, ). The droplet digital PCR has been used for many animal and human viruses such as Japanese encephalitis virus (Wu et al., ), porcine reproductive and respiratory syndrome virus (Yang et al., ), orthopoxviruses (Americo, Earl, & Moss, ), Merkel cell polyomavirus (Arvia et al., ), infectious hematopoietic necrosis virus (Jia et al., ), human papilloma virus (Lillsunde Larsson & Helenius, ), human immunodeficiency virus (Dunay et al., ), hepatitis E virus (Nicot et al., ) and human cytomegalovirus (Sedlak, Cook, Cheng, Magaret, & Jerome, ).…”

Section: Introductionmentioning

confidence: 99%

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples

Pinheiro‐de‐Oliveira

Fonseca-Júnior

Camargos

et al. 2019

Transbound Emerg Dis

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Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot‐and‐mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT‐ddPCR) using one‐step and two‐step approaches. Analytical sensitivity and specificity were done in parallel with real‐time PCR, RT‐qPCR (one‐step and two‐step) for comparison of sensitivity and specificity of both methods. In the standardization of RT‐ddPCR, the double‐quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one‐step techniques showed superior performance than two‐step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT‐ddPCR and RT‐qPCR using the one‐step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT‐PCR had a better performance than RT‐PCR when swine serum pools were tested. According to the results, the one‐step RT‐ddPCR and RT‐qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT‐ddPCR), being a useful tool for vesicular diseases control programs.

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Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

Cited by 17 publications

References 37 publications

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

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

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples

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