ABSTRACT
Background: Supply chain disruptions during the COVID-19 pandemic have affected the availability of components for specimen collection kits to detect SARS-CoV-2. Plastic injection molding offers a rapid and cheap method for mass production of swabs for upper respiratory tract sampling. Local production of virus transport medium increases flexibility to assemble sample collection kits if the medium provides appropriate stability for SARS-CoV-2 detection. Methods: A locally produced virus transport medium and a novel injection molded plastic swab were validated for SARS-CoV-2 detection by reverse-transcription quantitative polymerase chain reaction. Both components were compared to standard counterparts using viral reference material and representative patient samples. Results: Clinical testing showed no significant differences between molded and flocked swabs. Commercial and in-house virus transport media provided stable test results for over 40 days of specimen storage and showed no differences in test results using patient samples. Conclusions: This collection kit provides new supply chain options for SARS-CoV-2 testing.
Subject(s)
COVID-19 , Neoplasms , Humans , SARS-CoV-2 , COVID-19 Testing , Pandemics , Nasopharynx/chemistry , Specimen Handling/methods , Culture Media , RNA, ViralABSTRACT
Whole genome sequencing (WGS) is considered the best instrument to track both virus evolution and the spread of new, emerging variants. However, WGS still does not allow the analysis of as many samples as qPCR does. Epidemiological and clinical research needs to develop advanced qPCR methods to identify emerging variants of SARS-CoV-2 while collecting data on their spreading in a faster and cheaper way, which is critical for introducing public health measures. This study aimed at designing a one-step RT-qPCR assay for multiplex detection of the Omicron lineage and providing additional data on its subvariants in clinical samples. The RT-qPCR assay demonstrated high sensitivity and specificity on multiple SARS-CoV-2 variants and was cross-validated by WGS.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , SARS-CoV-2/genetics , Biological Assay , Public HealthABSTRACT
The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Humans , Reagent Kits, DiagnosticABSTRACT
The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein, as well as the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients, needs further longitudinal investigation. Several new serological methods to examine these parameters were developed, validated and applied in three patients of a family which underwent an ambulatory course of COVID-19 for six months. The virus load had almost completely disappeared after about four weeks. Serum IgA levels to the S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau at approximately six weeks, and stayed elevated over the observation period. Virus-neutralizing activity reached a peak about 4 weeks after disease onset but dropped slowly. The longitudinal associations of virus neutralization and the serological immune response suggest immunity in patients even after a mild clinical course of COVID-19.