Internal Validation of a Real-Time qPCR Kit following the UNE/EN ISO/IEC 17025:2005 for Detection of the Re-Emerging Monkeypox virus

Martínez‐Murcia, Antonio; Navarro, Aaron; Garcia-Sirera, Adrian; Pérez, Laura; Bru, Gema

doi:10.3390/diagnostics13091560

Cited by 2 publications

(1 citation statement)

References 33 publications

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“…We evaluated the efficiency and analytical sensitivity of the entire process starting from the sample and achieved a LOD of 10 copies/qPCR (95% confidence interval, CI), which is comparable to the LOD (~15.8 copies/PCR) reported by the CDC ( 5 ). Process efficiencies for N1 and N2 assays are well within an acceptable range ( 59 , 60 ). Our analysis also included multiple real-time PCR instruments and two different master mixes (TP-MM and TM-MM) to provide much-needed flexibility while maintaining the same analytical sensitivity and similar efficiency, demonstrating the robustness of the process and assay performance.…”

Section: Discussionmentioning

confidence: 80%

A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples

Millward,

Popelka,

Gutierrez

et al. 2023

Front. Public Health

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At the peak of the COVID-19 pandemic, pooled surveillance strategies were employed to alleviate the overwhelming demand for clinical testing facilities. A major drawback of most pooled-testing methods is the dilution of positive samples, which leads to a loss of detection sensitivity and the potential for false negatives. We developed a novel pooling strategy that compensates for the initial dilution with an appropriate concentration during nucleic acid extraction and real-time PCR. We demonstrated the proof of principle using laboratory-created 10-sample pools with one positive and corresponding individual positive samples by spiking a known amount of heat-inactivated SARS-CoV-2 into viral transport medium (VTM) or pooled negative saliva. No Ct difference was observed between a 10-sample pool with one positive vs. the corresponding individually analyzed positive sample by this method, suggesting that there is no detectable loss of sensitivity. We further validated this approach by using nasopharyngeal swab (NPS) specimens and showed that there is no loss of sensitivity. Serial dilutions of the virus were spiked into VTM and pooled with negative saliva in simulated 10-sample pools containing one positive to determine the LOD and process efficiency of this pooling methodology. The LOD of this approach was 10 copies/PCR, and the process efficiencies are ~95%−103% for N1 and ~87%−98% for N2 with samples in different matrices and with two different master mixes tested. Relative to TaqPath 1-step master mix, the TaqMan Fast Virus 1-Step master mix showed better sensitivity for the N2 assay, while the N1 assay showed no Ct difference. Our pooled testing strategy can facilitate large-scale, cost-effective SARS-CoV-2 surveillance screening and maintain the same level of sensitivity when analyzed individually or in a pool. This approach is highly relevant for public health surveillance efforts aimed at mitigating SARS-CoV-2 spread.

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Section: Discussionmentioning

confidence: 80%

A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples

Millward,

Popelka,

Gutierrez

et al. 2023

Front. Public Health

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Review of virological methods for laboratory diagnosis and characterization of monkeypox virus (MPXV): lessons learned from the 2022 Mpox outbreak

Resman Rus,

Zakotnik,

Sagadin

et al. 2024

Acta Dermatovenerologica Alpina Pannonica Et Adriatica

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Monkeypox virus (MPXV), originally endemic in West Africa (Clade II) and Central Africa (Clade I), has recently emerged worldwide and has reinforced the need for rapid and accurate MPXV diagnostics. This review presents and critically discusses the range of virological methods for laboratory diagnosis and characterization of MPXV as well as related lessons learned and practical experience gained from the 2022 Mpox global outbreak. Real-time PCR is currently considered the diagnostic gold standard and ensures accurate and timely confirmation of suspected Mpox cases based on suspicious skin lesions, and digital PCR improves the precision of MPXV DNA quantification. Whole genome sequencing reveals the diversity within the Clade IIb outbreak and highlights the role of microevolution in the adaptation of the virus to the human host. Continuous genomic surveillance is important for better understanding of human-to-human transmission and prevention of the emergence of variola virus-like strains. Traditional virological methods such as electron microscopy and virus isolation remain essential for comprehensive virus characterization, particularly in the context of vaccine and antiviral drug development. Despite the current challenges, serological tests detecting a range of anti-MPXV antibodies are important adjunct diagnostic and research tools for confirmation of late-presenting or asymptomatic MPXV cases, contact tracing, epidemiological studies, seroepidemiological surveys, and better understanding of the role of IgG and neutralizing antibodies in the immune response to infection and vaccination. A multidisciplinary approach combining advanced molecular techniques with traditional virological methods is important for rapid and reliable diagnosis, surveillance, and control of the outbreak.

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Internal Validation of a Real-Time qPCR Kit following the UNE/EN ISO/IEC 17025:2005 for Detection of the Re-Emerging Monkeypox virus

Cited by 2 publications

References 33 publications

A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples

A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples

Review of virological methods for laboratory diagnosis and characterization of monkeypox virus (MPXV): lessons learned from the 2022 Mpox outbreak

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