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17 pages, 2897 KiB

Open AccessArticle

Development and Testing of a Low-Cost Inactivation Buffer That Allows for Direct SARS-CoV-2 Detection in Saliva

by Brandon Bustos-Garcia, Sylvia Garza-Manero, Nallely Cano-Dominguez, Dulce Maria Lopez-Sanchez, Gonzalo Salgado-Montes de Oca, Alfonso Salgado-Aguayo, Felix Recillas-Targa, Santiago Avila-Rios and Victor Julian Valdes

Vaccines 2022, 10(5), 730; https://doi.org/10.3390/vaccines10050730 - 6 May 2022

Cited by 3 | Viewed by 2886

Abstract

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing. Full article

(This article belongs to the Special Issue The COVID Vaccine)

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12 pages, 805 KiB

Open AccessArticle

Comparative Analysis of Five Multiplex RT-PCR Assays in the Screening of SARS-CoV-2 Variants

by Vanessa De Pace, Bianca Bruzzone, Andrea Orsi, Valentina Ricucci, Alexander Domnich, Giulia Guarona, Nadia Randazzo, Federica Stefanelli, Enrico Battolla, Pier Andrea Dusi, Flavia Lillo and Giancarlo Icardi

Microorganisms 2022, 10(2), 306; https://doi.org/10.3390/microorganisms10020306 - 27 Jan 2022

Cited by 20 | Viewed by 4846

Abstract

The rapid and presumptive detection of SARS-CoV-2 variants may be performed using multiplex RT-PCR assays. The aim of this study was to evaluate the diagnostic performance of five qualitative RT-PCR tests as compared with next-generation sequencing (NGS). We retrospectively examined a multi-variant panel (n = 72) of SARS-CoV-2-positive nasopharyngeal swabs categorized as variants of concern (Alpha, Beta, Gamma and Delta), variants under monitoring (Iota and Kappa) and wild-type strains circulating in Liguria (Italy) from January to August 2021. First, NGS libraries of study samples were prepared and mapped to the reference genome. Then, specimens were screened for the detection of L452R, W152C, K417T, K417N, E484Q, E484K and N501Y mutations using the SARS-CoV-2 Variants II Assay Allplex, UltraGene Assay SARS-CoV-2 452R & 484K & 484Q Mutations V1, COVID-19 Ultra Variant Catcher, SARS-CoV-2 Extended ELITe MGB and Simplexa SARS-CoV-2 Variants Direct. The overall accuracy of these assays ranged from 96.9% to 100%. Specificity and sensitivity were 100% and 96–100%, respectively. We highly recommend the use of these assays as second-level tests in the routine workflow of SARS-CoV-2 laboratory diagnostics, as they are accurate, user friendly, low cost, may identify specific mutations in about 2–3 h and, therefore, optimize the surveillance of SARS-CoV-2 variants. Full article

(This article belongs to the Special Issue Rapid and Novel Diagnostics for Infectious Diseases)

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16 pages, 7470 KiB

Open AccessArticle

Interactome Analysis of iPSC Secretome and Its Effect on Macrophages In Vitro

by Luca Tamò, Kleanthis Fytianos, Fabienne Caldana, Cedric Simillion, Anis Feki, Izabela Nita, Manfred Heller, Thomas Geiser and Amiq Gazdhar

Int. J. Mol. Sci. 2021, 22(2), 958; https://doi.org/10.3390/ijms22020958 - 19 Jan 2021

Cited by 7 | Viewed by 4502

Abstract

Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration. Full article

(This article belongs to the Special Issue hiPSC-Derived Cells as Models for Drug Discovery)

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10 pages, 1576 KiB

Open AccessCommunication

A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity

by Sara Petrillo, Giovanna Carrà, Paolo Bottino, Elisa Zanotto, Maria Chiara De Santis, Jean Piero Margaria, Alessandro Giorgio, Giorgia Mandili, Miriam Martini, Rossana Cavallo, Davide Barberio and Fiorella Altruda

Microorganisms 2020, 8(7), 1064; https://doi.org/10.3390/microorganisms8071064 - 17 Jul 2020

Cited by 40 | Viewed by 7242

Abstract

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed. Full article

(This article belongs to the Special Issue COVID-19: Focusing on Epidemiologic, Virologic, and Clinical Studies)

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