Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective

Hossain, Md. Walid; Hossain, M. Pear; Arafath, Khalid; Ety, Subarna Sayed; Shetu, Md. Mahade Hasan; Kabir, Mazbahul; Noor, Farjana Akther; Mannoor, Kaiissar

doi:10.1371/journal.pone.0276464

Cited by 7 publications

(3 citation statements)

References 14 publications

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“…This lower linearity could be attributed to the “ultra-rapid” thermal cycling utilized by PicoGene ( 18 – 20 ). Even the “fast mode” of the conventional real-time PCR instrument showed a tendency toward lower linearity under fast PCR conditions ( 27 , 28 ). However, despite the lower linearity, the limit of detection achieved by PicoGene with the CDC N1/N2 duplex assay was 1 copy/µL ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Rapid and sensitive on-site detection of SARS-CoV-2 RNA from environmental surfaces using portable laboratory devices

Kitamura,

Ueno,

Yoshida

2023

Microbiol Spectr

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Monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on inanimate surfaces plays a crucial role in environmental surveillance, enabling the indirect detection of infected individuals and facilitating rapid infection control responses. However, developing a practical on-site, end-to-end method for detection remains a challenge. In this study, we established protocols for on-site detection of SARS-CoV-2 RNA on surfaces using two portable laboratory devices: The PicoGene PCR1100 and Bento Lab. Test particles comprised of inactivated viral particles containing partial sequences of SARS-CoV-2, type 1 poliovirus, coxsackie virus A16, and norovirus genogroups I and II were applied to reference materials as simulated contaminants. The direct detection method with the PicoGene PCR1100 achieved a recovery efficiency of 35.1% for the dried reference material. Furthermore, the Bento Lab, equipped with a microcentrifuge for on-site RNA purification, successfully detected a low concentration of inactivated SARS-CoV-2 reference standard. The recovery efficiency of the on-site RNA purification method on plastic, stainless steel, glass, and polyvinyl chloride surfaces ranged from 15.5% to 21.2%. Through the combination of these portable devices, SARS-CoV-2 RNA was detected within 1 h of swab sampling. This study contributes to the on-site risk assessment of infectious diseases by enabling the rapid detection of viral genomes. IMPORTANCE This study presents the development of a highly sensitive on-site method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on various surfaces, including doorknobs and tables. Identifying SARS-CoV-2 RNA on these surfaces can be crucial in guiding decision-making for implementing non-pharmaceutical interventions, such as zoning strategies, improving ventilation, maintaining physical distancing, and promoting increased hand hygiene practices. Moreover, the on-site detection system can facilitate the swift initiation of mitigation responses in non-laboratory settings, including long-term care facilities and schools. The protocols established in this study offer a comprehensive approach for achieving both sensitivity and rapidity in on-site SARS-CoV-2 RNA detection. Furthermore, since the RT-qPCR assay serves as the gold standard for detecting viral RNAs, the developed protocol holds potential for application to other viruses, including enteroviruses and noroviruses.

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Section: Discussionmentioning

confidence: 99%

Rapid and sensitive on-site detection of SARS-CoV-2 RNA from environmental surfaces using portable laboratory devices

Kitamura,

Ueno,

Yoshida

2023

Microbiol Spectr

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“…But some of the mutations acquired during the evolution of the viral genome may lead to failure in RT-PCR amplification, for instance the 69-70 del in the S gene [ 9 , 10 ] as well as mutations occurring in the N gene [ 11 , 12 ]. Globally, this technique is considered the gold standard for the diagnosis of COVID-19 due to its sensitivity; therefore, many industries had developed commercial kits to reduce the typical run time and improve the methodology protocol [ 13 , 14 , 15 ]. The importance of a solid and proven stability of genomic regions is pivotal to design targets for a diagnostic kit, therefore in this study we compared 48 samples identified during the daily diagnostic workflow presenting a PCR peculiar result.…”

Section: Introductionmentioning

confidence: 99%

Genomic and Temporal Analysis of Deletions Correlated to qRT-PCR Dropout in N Gene in Alpha, Delta and Omicron Variants

Gatti,

Brandolini,

Mancini

et al. 2023

Viruses

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Since the first SARS-CoV-2 outbreak, mutations such as single nucleotide polymorphisms (SNPs) and insertion/deletions (INDELs) have changed and characterized the viral genome sequence, structure and protein folding leading to the onset of new variants. The presence of those alterations challenges not only the clinical field but also the diagnostic demand due to failures in gene detection or incompleteness of polymerase chain reaction (PCR) results. In particular, the analysis of understudied genes such as N and the investigation through whole-genome next generation sequencing (WG-NGS) of regions more prone to mutate can help in the identification of new or reacquired mutations, with the aim of designing robust and long-lasting primers. In 48 samples of SARS-CoV-2 (including Alpha, Delta and Omicron variants), a lack of N gene amplification was observed in the genomes analyzed through WG-NGS. Three gene regions were detected hosting the highest number of SNPs and INDELs. In several cases, the latter can interfere deeply with both the sensitivity of diagnostic methodologies and the final protein folding. The monitoring over time of the viral evolution and the reacquisition among different variants of the same mutations or different alterations within the same genomic positions can be relevant to avoid unnecessary consumption of resources.

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“…However, the time required is longer than that for other tests, owing to the time required for nucleic acid extraction and amplification [ 17 ]. Therefore, studies have aimed to shorten the assay time by focusing on pooled testing, nucleic acid extraction, and PCR amplification [ 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Rapid Multiplex Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for SARS-CoV-2 Detection in Individual and Pooled Samples

Baek,

Park,

Lim

et al. 2023

Life

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Although coronavirus disease 2019 (COVID-19) is no longer a Public Health Emergency of International Concern (PHEIC), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a vast impact to date. Hence, continuous management is required, given the uncertainty caused by the potential evolution of SARS-CoV-2. Reverse transcription-quantitative PCR (RT-qPCR) diagnosis has been fundamental in overcoming this issue. In this study, the performances of two rapid RT-qPCR assays (Real-Q Direct SARS-CoV-2 Detection Kit and Allplex™ SARS-CoV-2 fast PCR Assay) with short PCR times were comparatively evaluated using a STANDARD M nCoV Real-Time Detection Kit (STANDARD M, conventional RT-qPCR assay). All kits showed a limit of detection values (102–103 copies/reaction). The evaluation showed that the two rapid assay tests had ≥97.89% sensitivity and ≥99.51% specificity (κ = 0.98) for individual samples and ≥97.32% sensitivity and ≥97.67% specificity for pooled samples compared to STANDARD M. These results indicate that the two rapid RT-qPCR kits, which showed significant time reduction in performance, are as effective as a conventional RT-qPCR assay. They are likely to increase not only the number of tests that can be performed but also the efficiency of sustainable management of COVID-19 in the long term.

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Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective

Cited by 7 publications

References 14 publications

Rapid and sensitive on-site detection of SARS-CoV-2 RNA from environmental surfaces using portable laboratory devices

Rapid and sensitive on-site detection of SARS-CoV-2 RNA from environmental surfaces using portable laboratory devices

Genomic and Temporal Analysis of Deletions Correlated to qRT-PCR Dropout in N Gene in Alpha, Delta and Omicron Variants

Evaluation of Rapid Multiplex Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for SARS-CoV-2 Detection in Individual and Pooled Samples

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