'CoviSwiftTM': A point-of-care RT-PCR device for SARS-CoV-2 and its variant detection.

We present the point-of-care (POC) molecular diagnostic solution, evaluated during COVID-19 pandemic caused by SARS-CoV-2 (Dec 2021). The POC comprised of a complete platform from self-sampling to RT-PCR testing of SARS-CoV-2 and its variants on portable Compact Q Real time polymerase chain reaction system. The multiplex assay was designed to target S, ORF1, and N genes of SARS-CoV-2 genome in a single tube with RNaseP as endogenous internal control. The present POC enables high accuracy (>95%) and high-throughput testing with a turnaround time of 45 min. It provides a unique platform from self-sample collection to report generation with rapid protocol, pipette and expert-free operation, long shelf-life stability and room temperature storage which enable to increase the efficiency of molecular testing. This novel test named "CoviSwift™ COVID-19 S PLUS RAPID PCR KIT" has been approved by CDSCO, Indian National Regulatory Authority, India, and is in use for clinical settings in India.

A Study of the Detection of SARS-CoV-2 by the Use of Electrochemiluminescent Biosensor Based on Asymmetric Polymerase Chain Reaction Amplification Strategy.

A new and reliable method has been constructed for detecting severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) open reading frames 1ab (ORF1ab) gene via highly sensitive electrochemiluminescence (ECL) biosensor technology based on highly efficient asymmetric polymerase chain reaction (asymmetric PCR) amplification strategy. This method uses magnetic particles coupled with biotin-labeled one complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab gene as the magnetic capture probes, and [Formula: see text]-labeled amino-modified another complementary nucleic acid sequence as the luminescent probes, and then a detection model of magnetic capture probes-asymmetric PCR amplification nucleic acid products-[Formula: see text]-labeled luminescent probes is formed, which combines the advantages of highly efficient asymmetric PCR amplification strategy and highly sensitive ECL biosensor technology, enhancing the method sensitivity of detecting the SARS-CoV-2 ORF1ab gene. The method enables the rapid and sensitive detection of the ORF1ab gene and has a linear range of 1-[Formula: see text] copies/[Formula: see text], a regression equation of [Formula: see text] = [Formula: see text] + 2919.301 ([Formula: see text] = 0.9983, [Formula: see text] = 7), and a limit of detection (LOD) of 1 copy/[Formula: see text]. In summary, it can meet the analytical requirements for simulated saliva and urine samples and has the benefits of easy operation, reasonable reproducibility, high sensitivity, and anti-interference abilities, which can provide a reference for developing efficient field detection methods for SARS-CoV-2.

Au Nanoparticles/HfO2/Fully Depleted Silicon-on-Insulator MOSFET Enabled Rapid Detection of Zeptomole COVID-19 Gene With Electrostatic Enrichment Process.

In this work, a novel sensing structure based on Au nanoparticles/HfO2/fully depleted silicon-on-insulator (AuNPs/HfO2/FDSOI) MOSFET is fabricated. Using such a planar double gate MOSFET, the electrostatic enrichment (ESE) process is proposed for the ultrasensitive and rapid detection of the coronavirus disease 2019 (COVID-19) ORF1ab gene. The back-gate (BG) bias can induce the required electric field that enables the ESE process in the testing liquid analyte with indirect contact with the top-Si layer. It is revealed that the ESE process can rapidly and effectively accumulate ORF1ab genes close to the HfO2 surface, which can significantly change the MOSFET threshold voltage ([Formula: see text]). The proposed MOSFET successfully demonstrates the detection of zeptomole (zM) COVID-19 ORF1ab gene with an ultralow detection limit down to 67 zM (~0.04 copy/[Formula: see text]) for a test time of less than 15 min even in a high ionic-strength solution. Besides, the quantitative dependence of [Formula: see text] variation on COVID-19 ORF1ab gene concentration from 200 zM to 100 femtomole is also revealed, which is further confirmed by TCAD simulation.

Ultrasensitive and amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor powered by CRISPR/Cas13a.

This study proposed a CRISPR/Cas13a-powered electrochemical multiplexed biosensor for detecting SARS-CoV-2 RNA strands. Current SARS-CoV-2 diagnostic methods, such as reverse transcription PCR (RT-PCR), are primarily based on nucleic acid amplification (NAA) and reverse transcription (RT) processes, which have been linked to significant issues such as cross-contamination and long turnaround times. Using a CRISPR/Cas13a system integrated onto an electrochemical biosensor, we present a multiplexed and NAA-free strategy for detecting SARS-CoV-2 RNA fragments. SARS-CoV-2 S and Orf1ab genes were detected in both synthetic and clinical samples. The CRISPR/Cas13a-powered biosensor achieved low detection limits of 2.5 and 4.5 ag/µL for the S and Orf1ab genes, respectively, successfully meeting the sensitivity requirement. Furthermore, the biosensor's specificity, simplicity, and universality may position it as a potential rival to RT-PCR.

Evaluation of CCR5-Î"32 mutation and HIV-1 surveillance drug-resistance mutations in peripheral blood mononuclear cells of long-term non progressors of HIV-1-infected individuals

Aim: This study aimed to evaluate chemokine receptor 5 delta 32 (CCR5-δ32) mutation and HIV-1 surveillance drug-resistance mutations (SDRMs) in peripheral blood mononuclear cells of long-term non progressors (LTNPs) of HIV-1-infected individuals. Materials & methods: This research was performed on 197 treatment-naive HIV-1-infected patients. After follow-up, it was determined that 15 (7.6%) of these people were LTNPs. The PCR assay was performed to identify the CCR5 genotype and HIV-1 SDRMs. Results: One (6.7%) of the LTNPs was heterozygous (wt/δ32) for the CCR5 delta 32 (CCR5δ32). However, none of the individuals was homozygous for this mutation (δ32/δ32). Moreover, none of the LTNPs showed HIV-1 SDRMs. The CRF35-AD subtype was the most dominant subtype, with a percentage of 93.3%. Conclusion: Iranian elite controllers are negative for CCR5-delta 32 homozygous genotype and drug resistance against antiretroviral drugs.

Performance evaluation of antigen detection rapid diagnostic test (Ag-RDT) for COVID-19 diagnosis in a primary healthcare center during the Shanghai COVID-19 quarantine period.

BACKGROUND: Rapid and accurate detection of SARS-CoV-2 infection is the cornerstone of prompt patient care. However, the reliability of the antigen rapid diagnostic test (Ag-RDT) in the diagnosis of SARS-CoV-2 infection remains inconclusive. METHODS: We conducted a field evaluation of Ag-RDT performance during the Shanghai Coronavirus disease 2019 (COVID-19) quarantine and screened 7225 individuals visiting our Emergency Department. 83 asymptomatic SARS-CoV-2 (+) individuals were enrolled in the current study. Simultaneously, Ag-RDT was performed to evaluate its testing performance. RESULTS: For the Ag-RDT(-) cases, the average cycle threshold (Ct) values of the N gene were 27.26 ± 4.59, which were significantly higher than the Ct value (21.9 ± 4.73) of the Ag-RDT(+) individuals (p < 0.0001). The overall sensitivity of Ag-RDT versus that of RT-PCR was 43.37%. The Ag-RDT(+) individuals regarding the N gene's Ct value were 16 cases in the < 20 range, 12 in 20-25, 5 in 25-30, and 3 in 30-35. The corresponding sensitivity was 84.21%, 52.17%, 21.74% and 16.67%, respectively. Meanwhile, sampling had a straight specificity of 100% regardless of the Ct value. CONCLUSIONS: The Ag-RDT were extremely sensitive in asymptomatic COVID-19 individuals with a Ct value < 20.

Establishment of a Duplex Real Time PCR Method for Detection of SARS-COV-2

To quickly and efficiently detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and prevent and control the spread of novel coronavirus disease (COVID-19), a highly sensitive duplex real-time PCR (RT-PCR) detection method has been established. In this study, the specificity of primers and probes were designed, respectively, according to the ORF1ab gene and N gene sequence of SARS-COV-2, and fluorescent probes were labeled with carboxyl fluorescein (FAM) and green fluorescent protein (VIC). The duplex RT-PCR method for detecting SARS-COV-2 with TaqMan probe was established, which has a limit of detection of 10 copies/µL, and the linear detection range of ORF1ab and N gene were 1.0 × 101-1.0 × 105 copies/µL and 1.0 × 101-1.0 × 106 copies/µL, respectively, realizing the simultaneous detection of ORF1ab and N genes in simulated SARS-COV-2 samples. The method has high sensitivity, accurate quantification, simple operation, and cost-saving, which can be used for rapid and efficient quantitative detection of SARS-COV-2.

Partial ORF1ab Gene Target Failure with Omicron BA.2.12.1.

Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.

A Study of the Detection of SARS-CoV-2 ORF1ab Gene by the Use of Electrochemiluminescent Biosensor Based on Dual-Probe Hybridization.

To satisfy the need to develop highly sensitive methods for detecting the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and further enhance detection efficiency and capability, a new method was created for detecting SARS-CoV-2 of the open reading frames 1ab (ORF1ab) target gene by a electrochemiluminescence (ECL) biosensor based on dual-probe hybridization through the use of a detection model of "magnetic capture probes-targeted nucleic acids-Ru(bpy)32+ labeled signal probes". The detection model used magnetic particles coupled with a biotin-labeled complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab target gene as the magnetic capture probes and Ru(bpy)32+ labeled amino modified another complementary nucleic acid sequence as the signal probes, which combined the advantages of the highly specific dual-probe hybridization and highly sensitive ECL biosensor technology. In the range of 0.1 fM~10 µM, the method made possible rapid and sensitive detection of the ORF1ab gene of the SARS-CoV-2 within 30 min, and the limit of detection (LOD) was 0.1 fM. The method can also meet the analytical requirements for simulated samples such as saliva and urine with the definite advantages of a simple operation without nucleic acid amplification, high sensitivity, reasonable reproducibility, and anti-interference solid abilities, expounding a new way for efficient and sensitive detection of SARS-CoV-2.

Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile.

Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.

Reliable Diagnostics of SARS-CoV-2 Infections Using One- and Two-Gene Molecular Tests for a Viral RNA Detection-Results Questioning Previous Observations.

SARS-CoV-2 is a new virus from the Coronaviridae family and its rapid spread is now the most important medical problem worldwide. Currently used tests vary in the number and selection of SARS-CoV-2 target genes. Meanwhile, the choice of the appropriate target gene may be important in terms of a reliable detection of a viral RNA. As some researchers questioned the sensitivity of the monogenic VIASURE SARS-CoV-2 S gene Real Time PCR Detection Kit (CerTest Biotec, Zaragoza, Spain) in mid-2020, the aim of the study was to evaluate the usefulness of this kit, used along with the BD MAX™ System (Becton Dickinson, East Rutherford, NJ, USA), and compare the results with two-gene Bosphore Novel Coronavirus (2019-nCoV) Detection Kit v1 (Anatolia Diagnostics and Biotechnology Products Inc., Istanbul, Turkey). Both tests were carried out on 306 nasopharyngeal/oropharyngeal swabs. The consistent results (72 positive and 225 negative results found simultaneously in both kits) were obtained for 297 (97.1%) samples altogether, while discrepancies between the results of the evaluated tests were observed for nine (2.9%) specimens. There were no statistically significant differences between the method used and the frequency of positive results. Both tests, targeted at detecting one and two genes, are effective in SARS-CoV-2 RNA detection.

Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor.

Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/µL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.

Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID-19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS-CoV-2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E- and N1-gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID-19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for >2-fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID-19 detection and significantly reduce the turn-around time of reporting COVID-19 test results.