Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system

Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, M.; Henquell, Cécile; Bailly, Jean‐Luc; Peigue-Lafeuille, H.; Archimbaud, Christine

doi:10.1016/j.jviromet.2012.06.019

Cited by 22 publications

(23 citation statements)

References 32 publications

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“…The purpose of performing our assay in separate reactions is to achieve specific and simultaneous detection, without the risk of cross-contamination. Using this method, 100% sensitivity was obtained for the 3 viruses, and we observed no cross-reactions with any of the viruses analyzed; additionally, specificities with high values were achieved (EV: 96%, HSV1: 100%, and HSV2: 94%), similar to those reported with the end-point and other real-time techniques, and even higher than the specificity achieved with other commercial kits [7,11,[16][17][18][19][20][21] . These results indicate that the sequences used for the primers and probes are specific; in the case of EV, the primers allow the identification of all viruses grouped in the genus, as the target sequence is highly conserved among its members; in the case of HSV1 and HSV2, as the primers are aimed at 2 different targets, this protocol can achieve differential diagnosis between these 2 viruses, without observing the cross-reactions that have been reported in other studies that use different areas of the same target [22][23][24] .…”

supporting

confidence: 83%

“…The reproducibility of the assay was demonstrated by the consistency of the 3 reactions in 5 replicates with plasmid-positive controls. Their Ct values and the CV indicated that the data behaved homogeneously in each dilution, with values being located within an acceptable range (0.33-2.37%) for diagnostic purposes [11,21] . Unlike many proposed real-time techniques for the detection of the viruses studied here, our test used an internal control for each sample in each round of amplification, which enabled verification of the integrity of the sample and the presence of contamination.…”

mentioning

confidence: 93%

“…Sarquiz-Martínez et al treatment, reducing the length and cost of hospitalization [10,11] . However, it is difficult to simultaneously detect the large number of causative agents of CNS infections through a multiplex single assay.…”

mentioning

confidence: 99%

“…The LOD for the 3 viruses were all 10 copies/μL, with very similar Ct values of 36.89, 35.13, and 37.44 for HSV1, HSV2, and EV, respectively. In previous studies on EV, a range of 30-37 for the Ct values has been reported to determine that a sample is positive [8,11,25] ; however, these values can vary, as they depend on both the quality of the standard used and the viral load in the CSF. Similarly, the results obtained for the HSV1 and HSV2 amplifications showed the expected LOD; however, it has been frequently reported that samples with low viral loads yield Ct values of >38 [2,9,21] .…”

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confidence: 99%

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Differential Detection of Enterovirus and Herpes Simplex Virus in Cerebrospinal Fluid by Real-Time RT-PCR

et al. 2017

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Background: Enterovirus (EV) and herpes simplex virus 1 and 2 (HSV1 and HSV2) are the main etiologic agents of central nervous system infections. Early laboratory confirmation of these infections is performed by viral culture of the cerebrospinal fluid (CSF), or the detection of specific antibodies in serum (e.g., HSV). The sensitivity of viral culture ranges from 65 to 75%, with a recovery time varying from 3 to 10 days. Serological tests are faster and easy to carry out, but they exhibit cross-reactivity between HSV1 and HSV2. Although molecular techniques are more sensitive (sensitivity >95%), they are more expensive and highly susceptible to cross-contamination. Methods: A real-time RT-PCR for the detection of EV, HSV1, and HSV2 was compared with end-point nested PCR. Results: We tested 87 CSF samples of patients with a clinical diagnosis of viral meningitis or encephalitis. Fourteen samples were found to be positive by RT-PCR, but only 8 were positive by end-point PCR. The RT-PCR showed a specificity range of 94-100%, the negative predictive value was 100%, and the positive predictive value was 62, 100, and 28% for HSV1, HSV2, and EV, respectively. Conclusion: Real-time RT-PCR detected EV, HSV1, and HSV2 with a higher sensitivity and specificity than end-point nested RT-PCR.

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confidence: 83%

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confidence: 93%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Differential Detection of Enterovirus and Herpes Simplex Virus in Cerebrospinal Fluid by Real-Time RT-PCR

et al. 2017

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“…Nucleic acids were extracted from 200 ml supernatant or the whole cells and supernatant using a NucliSens EasyMAG extractor (bioMérieux) and were eluted with 25 ml elution buffer provided by the manufacturer. A previously described competitive internal control was added during the extraction step and amplified in our in-house qRT-PCR assay (Volle et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood–brain barrier

Volle

Archimbaud

Couraud

et al. 2015

Journal of General Virology

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Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.

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Evaluation of enterovirus nucleic acids detection method based on ultra‐fast real‐time fluorescence RT‐PCR technology—A pilot study

Gao

Hao

et al. 2022

Journal of Medical Virology

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The outbreak of COVID-19 epidemic has enabled the establishment and application of various rapid detection methods. It is particularly important to establish a fast and accurate detection method for enterovirus, which will be beneficial for clinical diagnosis, epidemic prevention and control, and timely traceability. Through establishing an ultra-fast reverse transcription-polymerase chain reaction (RT-PCR) equipment, this study aimed to evaluate the sensitivity and specificity of the testing method of enterovirus nucleic acids based on ultra-fast real-time fluorescence RT-PCR technology. A total of 61 cases were sampled, which were then transported and preserved. After the nucleic acid extraction, the nucleic acids of the same sample were tested with the enterovirus nucleic acid detection kit produced by Guangzhou Da An Gene Company and the ultra-fast RT-PCR equipment system established in this study. ABI7500Fast and Ahram biosystems S1 fast equipment were used for amplification detection. If the sample had an S-shaped amplification curve in the FAM channel and the Ct value ≤40.00, the result was positive. The sensitivity, precision, and accuracy of the detection method were then verified. This study established a novel testing method to achieve enterovirus nucleic acid detection within 24 min. The sensitivity detection limit of the method was 1.0 × 10 2 copies/ml. The coefficients of variation for repeated detection of the high, medium, and low concentration samples were 2.644%, 1.674%, and 4.281%, respectively, with good detection repeatability. In addition, a total of 29 cases were positive by the ultra-fast RT-PCR detection method in 61 suspected samples, which was consistent with the conventional fluorescent RT-PCR method. The established rapid detection method can greatly shorten the time for providing a detection report, which may greatly improve the efficiency of diagnosis and treatment.

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Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system

Cited by 22 publications

References 32 publications

Differential Detection of Enterovirus and Herpes Simplex Virus in Cerebrospinal Fluid by Real-Time RT-PCR

Differential Detection of Enterovirus and Herpes Simplex Virus in Cerebrospinal Fluid by Real-Time RT-PCR

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood–brain barrier

Evaluation of enterovirus nucleic acids detection method based on ultra‐fast real‐time fluorescence RT‐PCR technology—A pilot study

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