Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies

Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, K.

doi:10.1016/j.jviromet.2017.04.003

Cited by 43 publications

(31 citation statements)

References 31 publications

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“…In order to confirm HTS findings or confirm suspected sequence cross-lane, a set of specific rRT-PCR assays were applied to CSF with a sufficient initial specimen leftover to extract the viral genomes using the NucliSENS easyMAG (bioMérieux, Geneva, Switzerland). Such assays were performed when needed and available for adenovirus (ADV), Torque teno virus (TTV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), human pegivirus-1 (HPgV-1), and Human herpesvirus 7 (HHV-7) (Table S1) [16,17,18,19,20]. The rRT-PCR assays were performed using the one-step QuantiTect Probe RT-PCR Kit (Qiagen, Hombrechtikon, Switzerland) in a StepOne Plus instrument (Applied Biosystems, Rotkreuz, Switzerland) for the HPgV-1 assays or using the TaqMan™ Universal PCR Master Mix (ThermoFisher, Reinach, Switzerland) on a QuantStudio 5 instrument (Applied Biosystems) for the others assays.…”

Section: Methodsmentioning

confidence: 99%

Viral Sequences Detection by High-Throughput Sequencing in Cerebrospinal Fluid of Individuals with and without Central Nervous System Disease

Schibler

Brito

Zanella

et al. 2019

Genes

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: Meningitis, encephalitis, and myelitis are various forms of acute central nervous system (CNS) inflammation, which can coexist and lead to serious sequelae. Known aetiologies include infections and immune-mediated processes. Despite advances in clinical microbiology over the past decades, the cause of acute CNS inflammation remains unknown in approximately 50% of cases. High-throughput sequencing was performed to search for viral sequences in cerebrospinal fluid (CSF) samples collected from 26 patients considered to have acute CNS inflammation of unknown origin, and 10 patients with defined causes of CNS diseases. In order to better grasp the clinical significance of viral sequence data obtained in CSF, 30 patients without CNS disease who had a lumbar puncture performed during elective spinal anaesthesia were also analysed. One case of human astrovirus (HAstV)-MLB2-related meningitis and disseminated infection was identified. No other viral sequences that can easily be linked to CNS inflammation were detected. Viral sequences obtained in all patient groups are discussed. While some of them reflect harmless viral infections, others result from reagent or sample contamination, as well as index hopping. Altogether, this study highlights the potential of high-throughput sequencing in identifying previously unknown viral neuropathogens, as well as the interpretation issues related to its application in clinical microbiology.

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Section: Methodsmentioning

confidence: 99%

Viral Sequences Detection by High-Throughput Sequencing in Cerebrospinal Fluid of Individuals with and without Central Nervous System Disease

Schibler

Brito

Zanella

et al. 2019

Genes

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“…Different studies have demonstrated the higher sensitivity of ddPCR compared to RT-qPCR ( 19 , 20 ). At present, ddPCR is one of the most sensitive methods used for the detection of low amounts of targets, including circulating DNA, microRNAs, circulating mutations, rare copy number variants, low viral nucleic acid targets representing a promising technology for use in clinical practice and in public and environmental health ( 21 - 24 ).…”

Section: Discussionmentioning

confidence: 99%

Droplet digital PCR for the detection and monitoring of Legionella pneumophila

et al. 2020

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Legionella pneumophila (L. pneumophila) is a harmful pathogen often found in water systems. In hospitals, the absence of L. pneumophila in water systems is mandatory by law, therefore, frequent and effective monitoring of water is of fundamental importance. Molecular methods based on reverse transcription-quantitative polymerase chain reaction (RT-qPcR) have been proposed for the detection of L. pneumophila, however, the sensitivity and accuracy of these methods have not been validated yet. Therefore, it is important to evaluate other strategies able to overcome the limits of culture-based and RT-qPcR methods. On these bases, we compared the sensitivity and accuracy of droplet digital PcR (ddPcR) and RT-qPcR in water samples with known concentrations of L. pneumophila and in an in vitro model of water heat treatments. ddPcR showed a higher sensitivity rate and accuracy compared to RT-qPcR in detecting low bacterial load. In addition, ddPcR is not affected by the presence of fragmented dNA and showed higher accuracy than RT-qPcR in monitoring the efficacy of heat shock treatments. In conclusion, ddPcR represents an innovative strategy to effectively detect L. pneumophila in water samples. Thanks to its high robustness, ddPcR could be applied also for the detection of L. pneumophila in patients with suspected legionellosis.

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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%

“…One o f the disad vantages of quantitative PCR is the need for standard curves that require high-efficiency values when it is necessary to quantify target molecules (Arvia et al, 2017). The digital PCR is considered a precise and sensitive technique that does not require reference curves (Gullett & Nolte, 2015).…”

mentioning

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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Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies

Cited by 43 publications

References 31 publications

Viral Sequences Detection by High-Throughput Sequencing in Cerebrospinal Fluid of Individuals with and without Central Nervous System Disease

Viral Sequences Detection by High-Throughput Sequencing in Cerebrospinal Fluid of Individuals with and without Central Nervous System Disease

Droplet digital PCR for the detection and monitoring of Legionella pneumophila

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

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