Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA

Pinheiro‐de‐Oliveira, T. F.; Fonseca, Antônio Augusto; Camargos, Marcelo Fernandes; Laguardia-Nascimento, Mateus; Oliveira, Anapolino Macedo de; Cottorello, Ana Cláudia Pinto; Goés-Neto, Aristóteles; Barbosa-Stancioli, Edel Figueiredo

doi:10.1016/j.jviromet.2018.06.015

Cited by 21 publications

(16 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This similarity can occur, mainly when oligonucleotides as primer and probe are the same. Other studies also reported similar results of analytical sensitivity between ddPCR and RT‐qPCR to viruses like HIV‐1 (Henrich, Gallien, Li, Pereyra, & Kuritzkes, ), Enterovirus 71 (Lui & Tan, ) and BK virus (poliomavirus) (Bateman et al., ) and Foot‐and‐mouth disease virus (Pinheiro‐de‐Oliveira et al., ). Our results showed a high analytical sensitivity, detecting low doses of SVA (7.41 doses/ml or 10 0.87 TCID50/ml or 0.185 TCID50) and, when plasmid was used, the limit of detection was in the order of fg (0.1 fg).…”

Section: Discussionmentioning

confidence: 55%

“…Molecular methods such as conventional PCR, real‐time PCR, in situ hybridization technique‐RNAscope, nested‐PCR and massive parallel sequencing have been already available to be used in the detection of viral RNA of SVA (Bracht, O'Hearn, Fabian, Barrette, & Sayed, ; Dallagnol, Otonel, Leme, Alfieri, & Alfieri, ; Feronato et al., ; Fowler et al., ; Laguardia‐Nascimento et al., ; Pinheiro‐de‐Oliveira et al., ; Resende, Marthaler, & Vannucci, ; Vannucci et al., ). However, reverse transcriptase followed by droplet digital PCR (RT‐ddPCR) standardized in the present study offers a new methodology for detection and absolute quantification for Senecavirus A .…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, these samples were considered essential to define the threshold of RT‐ddPCR one‐step since a drop appeared in an amplitude below the positive control in 16% of the samples. The use of negative samples to better determine the cut‐off of the RT‐ddPCR was already established for FMDV (Pinheiro‐de‐Oliveira et al., ).…”

Section: Discussionmentioning

confidence: 99%

“…The partial VP1 gene of this virus was sequenced and deposited in GenBank database (KT007135) (Laguardia‐Nascimento et al., ). In addition, the serial dilutions of pUCIDT Kan plasmid (10 −1 to 10 −8 ; IDT, USA; 0.02 ng/μl) with kanamycin resistance gene containing the insertion of the partial 3D coding region SVA (78 bp) were also used to determine the limit of detection (Pinheiro‐de‐Oliveira et al., ). Raw data related to the analytical sensitivity of one‐step and two‐step techniques (RT‐ddPCR and RT‐qPCR) using virus and plasmid are presented in Table .…”

Section: Methodsmentioning

confidence: 99%

“…The analytical sensitivity was performed using serial dilutions (10 −1 to 10 −8 ) of Senecavirus A (10 6.87 TCID50/mL) isolated from swine epithelial tissue of Uberlândia (Minas Gerais, Brazil) during a Brazilian outbr e ak occurred in 2015. This virus was isolated in human lung cell l ine, free of contaminants and derived from a metastatic site (lymph node), NCI-H1299 (ATCC ® CRL-5803 ™ ), after the fifth passage (Pinheiro de Oliveira et al, 2013 serial dilutions of pUCIDT Kan plasmid (10 −1 to 10 −8 ; IDT, USA; 0.02 ng/μl) with kanamycin resistance gene containing the insertion of the partial 3D coding region SVA (78 bp) were also used to determine the limit of detection (Pinheiro- de-Oliveira et al, 2018). Raw data related to the analytical sensitivity of one-step and two-step techniques (RT-ddPCR and RT-qPCR) using virus and plasmid are presented in Table 3.…”

Section: Analytical Sensitivitymentioning

confidence: 99%

See 4 more Smart Citations

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

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Analytical Sensitivitymentioning

confidence: 99%

See 3 more Smart Citations

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

View full text Add to dashboard Cite

show abstract

Changes in peripheral blood cytokines in patients with severe fever with thrombocytopenia syndrome

Wang

et al. 2021

Journal of Medical Virology

View full text Add to dashboard Cite

Severe fever with thrombocytopenia syndrome (SFTS) is recognized as an emerging infectious disease. This study aimed to investigate the pathogenic mechanism of SFTS. A total of 100 subjects were randomly included in the study. Cytokine levels were detected by enzyme‐linked immunosorbent assay and the viral load was detected by micro drop digital PCR. The results showed that levels of interleukin‐6 (IL‐6), IL‐8, IL‐10, IFN‐inducible protein‐10 (IP‐10), monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), transforming growth factor‐β1 (TGF‐β1), and regulated upon activation normal T cell expressed and secreted factor (RANTES) differed significantly among the SFTS patient group, healthy people group, and asymptomatic infection group (p < .05). Compared to the healthy people group, the patient group had increased cytokine levels (IL‐6, IL‐10, IP‐10, MCP‐1, and IFN‐γ) but reduced levels of IL‐8, TGF‐β1, and RANTES (p < .0167). IL‐6, IL‐8, IL‐10, IP‐10, MCP‐1, MIP‐1α, TGF‐β1, and the RANTES levels had different trends after the onset of the disease. IL‐6, IL‐10, IP‐10, and MCP‐1 levels in severe patients were higher than those in mild patients (p < .05). There was a positive correlation between viral load and IL‐6 and IP‐10 but a negative correlation between viral load and RANTES. SFTSV could cause a cytokine change: the cytokine levels of patients had different degrees of fluctuation after the onset of the disease. The levels of IL‐6 and IL‐8 in the asymptomatic infection group were found between the SFTS patients group and the healthy people group. The levels of IL‐6, IL‐10, IP‐10, and MCP‐1 in the serum could reflect the severity of the disease, and the levels of IL‐6, IP‐10, and RANTES were correlated with the viral load.

show abstract

Development of quantitative PCR and digital PCR for the quantification of Leishmania infantum in dogs

et al. 2023

View full text Add to dashboard Cite

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA

Cited by 21 publications

References 25 publications

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

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

Changes in peripheral blood cytokines in patients with severe fever with thrombocytopenia syndrome

Development of quantitative PCR and digital PCR for the quantification of Leishmania infantum in dogs

Contact Info

Product

Resources

About