Utility of droplet digital PCR for the quantitative detection of polyomavirus JC in clinical samples

Giovannelli, Irene; Ciccone, Nunziata; Vaggelli, Guendalina; Malva, Nunzia Della; Torricelli, Francesca; Rossolini, Gian María; Giannecchini, Simone

doi:10.1016/j.jcv.2016.07.008

Cited by 13 publications

(4 citation statements)

References 21 publications

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“…The advantages of dPCR include absolute quantification without a calibration curve, high precision, and high accuracy even in the presence of inhibitors ( 5 ). dPCR and reverse transcription-dPCR have been used to determine the copy numbers of DNA and RNA viruses, such as human immunodeficiency virus (HIV), cytomegalovirus, hepatitis B virus, JC polyomavirus, human papillomavirus, human T-cell lymphotropic virus, and human rhinoviruses, in a variety of clinical specimens ( 7 – 12 , 17 , 18 ). Here, we employed dPCR to determine the RNA copy numbers of SARS-CoV-2 in the original nasopharyngeal swab samples and the inactivated samples and to evaluate the influence of different inactivation methods on the viral copy number.…”

Section: Discussionmentioning

confidence: 99%

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Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

Chen

Xing

et al. 2020

J Clin Microbiol

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The outbreak of COVID-19 has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed with three different methods: (1) incubation with TRIzol® LS Reagent for 10 min at room temperature, (2) heating in a waterbath at 56°C for 30 min, and (3) high-temperature treatment, including 121°C autoclaving for 20 min, 100°C boiling for 20 min, and 80°C heating for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of N gene and 39.85% of ORF 1ab. For samples treated at 56°C for 30 min, the copy number of N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. Viral RNA copy number dropped by 50–66% after 80°C heating for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicated that inactivation reduced the quantity of detectable viral RNA and may cause false negative results especially in weakly positive cases. Thus, TRIzol is recommended for sample inactivation in comparison to heat inactivation as Trizol has the least effect on RNA copy number among the tested methods.

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Section: Discussionmentioning

confidence: 99%

“…dPCR has many potential advantages over quantitative PCR, including the capability to obtain absolute quantification without external references and the tolerance of PCR inhibitors (6). dPCR is increasingly used in clinical virology for the study of human-pathogenic viruses (7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

Chen

Xing

et al. 2020

J Clin Microbiol

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“…Although different approaches to DNA library amplification were proposed (23,29,30), alternative methodologies are still in demand. The benefits of transition from bulk PCR to ePCR amplification come to the fore in droplet digital PCR, which outperforms classical DNA quantification techniques, such as qPCR (31), and is extensively used for absolute DNA quantification (32)(33)(34)(35)(36)(37)(38). Similarly, ePCR provides a reproducible DNA libraries amplification platform, outperforming conventional bulk PCR.…”

Section: Discussionmentioning

confidence: 99%

Liquid drop of DNA libraries reveals total genome information

Terekhov

Eliseev

Ovchinnikova

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

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“…The failure to detect JCPyV can postpone the diagnosis and, consequently, treatment options. The use of ddPCR to detect JCPyV DNA has also been recently described [27] and the authors further underline the high sensitivity of this technology to measure JC viral DNA. The use of highly sensitive and reliable PCR assays like ddPCR have also been reported in other clinical settings, such as SARS-CoV-2 infection [28][29][30].…”

Section: Discussionmentioning

confidence: 94%

Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy

Ngouth

Monaco

Walker

et al. 2022

Viruses

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Background: Lytic infection of oligodendrocytes by the human JC polyomavirus (JCPyV) results in the demyelinating disease called progressive multifocal leukoencephalopathy (PML). The detection of viral DNA in the cerebrospinal fluid (CSF) by PCR is an important diagnostic tool and, in conjunction with defined radiological and clinical features, can provide diagnosis of definite PML, avoiding the need for brain biopsy. The main aim of this study is to compare the droplet digital PCR (ddPCR) assay with the gold standard quantitative PCR (qPCR) for the quantification of JC viral loads in clinical samples. Methods: A total of 62 CSF samples from 31 patients with PML were analyzed to compare the qPCR gold standard technique with ddPCR to detect conserved viral DNA sequences in the JCPyV genome. As part of the validation process, ddPCR results were compared to qPCR data obtained in 42 different laboratories around the world. In addition, the characterization of a novel triplex ddPCR to detect viral DNA sequence from both prototype and archetype variants and a cellular housekeeping reference gene is described. Triplex ddPCR was used to analyze the serum from six PML patients and from three additional cohorts, including 20 healthy controls (HC), 20 patients with multiple sclerosis (MS) who had never been treated with natalizumab (no-NTZ-treated), and 14 patients with MS who were being treated with natalizumab (NTZ-treated); three from this last group seroconverted during the course of treatment with natalizumab. Results: JCPyV DNA was detected only by ddPCR for 5 of the 62 CSF samples (8%), while remaining undetected by qPCR. For nine CSF samples (15%), JCPyV DNA was at the lower limit of quantification for qPCR, set at <250 copies/mL, and therefore no relative quantitation could be determined. By contrast, exact copies of JCPyV for each of these samples were quantified by ddPCR. No differences were observed between qPCR and ddPCR when five standardized plasma samples were analyzed for JCPyV in 42 laboratories in the United States and Europe. JCPyV-DNA was undetected in all the sera from HC and MS cohorts tested by triplex ddPCR, while serum samples from six patients with PML tested positive for JCPyV. Conclusion: This study shows strong correlation between ddPCR and qPCR with increased sensitivity of the ddPCR assay. Further work will be needed to determine whether multiplex ddPCR can be useful to determine PML risk in natalizumab-treated MS patients.

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Utility of droplet digital PCR for the quantitative detection of polyomavirus JC in clinical samples

Cited by 13 publications

References 21 publications

Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

Liquid drop of DNA libraries reveals total genome information

Comparison of qPCR with ddPCR for the Quantification of JC Polyomavirus in CSF from Patients with Progressive Multifocal Leukoencephalopathy

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