Quadruplex Droplet Digital PCR Assay for Screening and Quantification of SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by the rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since early 2020, has highlighted the need for sensitive and reliable diagnostic methods. Droplet digital PCR (ddPCR) has demonstrated superior performance over the gold-standard reverse transcription PCR (RT-PCR) in detecting SARS-CoV-2. In this study, we explored the development of a multiplex ddPCR assay that enables sensitive quantification of SARS-CoV-2, which could be utilized for antiviral screening and the monitoring of COVID-19 patients. We designed a quadruplex ddPCR assay targeting four SARS-CoV-2 genes and evaluated its performance in terms of specificity, sensitivity, linearity, reproducibility, and precision using a two-color ddPCR detection system. The results showed that the quadruplex assay had comparable limits of detection and accuracy to the simplex ddPCR assays. Importantly, the quadruplex assay demonstrated significantly improved performance for samples with low viral loads and ambiguous results compared to the standard qRT-PCR approach. The developed multiplex ddPCR represents a valuable alternative and complementary tool for the diagnosis of SARS-CoV-2 and potentially other pathogens in various application scenarios beyond the current COVID-19 pandemic. The improved sensitivity and reliability of this assay could contribute to more effective disease monitoring and antiviral screening during the ongoing public health crisis.

Diagnosis with Metagenomic Next-Generation Sequencing (mNGS) technology and real-time PCR for SARS-CoV-2 Omicron detection using various nasopharyngeal swabs in SARS-CoV-2 Omicron.

BACKGROUND: The SARS-CoV-2 Omicron variant, with the main subtypes BA.5.2 and BF.7 in China, led to off-target effects on the S and N genes from December 1, 2022, to January 31, 2023. The kits used for studying and developing these agents were not adequately and independently evaluated. It is important to verify the performance of commercial Real-Time quantitative PCR (RT-qPCR) tests. OBJECTIVE: We conducted a clinical evaluation of two Real Time SARS-CoV-2 Omicron assays to verify their performance using various detection reagents and clinical specimens. METHODS: We performed clinical evaluations of two existing Chinese SARS-CoV-2 Omicron RT-qPCR kits 2019-nCoV nucleic acid diagnostic kits (Fosun Biotechnology, National instrument registration 20203400299, Shanghai, China) and COVID-19 nucleic acid detection kits (eDiagnosis Biomedicine, National instrument registration 20203400212, Wuhan, China) and using BSD (Bondson) (Guangzhou Bondson Biotechnology Co. Ltd, batch number 2022101), quality controls provided by the inspection center and a large number of clinically confirmed specimens. RESULTS: The concordance rates for the Fosun and eDiagnosis kits were 95% and 100%, respectively. The detection limit for the Fosun and eDiagnosis kits was verified to be 300 copies/mL and 500 copies/mL. The Fosun assay exhibited the largest coefficient of variation (CV) for ORF1ab and N gene at the detection limit concentration (4.80%, 3.49%), whereas eDiagnosis showed a smaller CV (0.93%, 1.10%). In the reference product from the Hangzhou Clinical Laboratory Center test, it was found that Fosun had the lowest sensitivity of 93.47% and a specificity of 100%, while eDiagnosis exhibited 100% for both sensitivity and specificity. The lowest single target gene detection rate of Fosun reagents was 68.7% for the ORF1ab gene and 87.5% for the N gene, while eDiagnosis detection rate was 100%. Among the clinical group S specimens, the missed detection rate of the Fosun reagent was 10.9%, which was higher than the 3.9% of eDiagnosis. However, there was no significant difference in the clinical diagnostic efficiency of the two reagents. CONCLUSIONS: The ORF1ab and N assays of SARS-CoV-2 Omicron on the eDiagnosis platform yielded higher values compared to those on the Fosun platform. Consequently, the eDiagnosis kit has also been used as standard detection reagents. Considering that the Fosun reagent has a relatively low detection limit and targets three single genes, it is more advantageous as a confirmatory reagent for the new museum.

Use of strips of rapid diagnostic tests as a source of ribonucleic acid for genomic surveillance of viruses: an example of SARS-CoV-2.

BACKGROUND: This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes. METHOD: It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested. RESULT: Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%. CONCLUSION: These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2.

Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Saliva Is a Sensitive and Accessible Sample Both for SARS-CoV-2 Detection and for the Evaluation of Treatment Effectiveness in Follow-Up Studies.

Despite emerging evidence indicating that molecular SARS-CoV-2 tests performed on saliva have diagnostic sensitivity and specificity comparable to those observed with nasopharyngeal swabs (NPSs), most in vivo follow-up studies on the efficacy of drugs against SARS-CoV-2 have been performed on NPSs, not considering saliva as a possible alternative matrix. For this reason, in this study, we used, in parallel, saliva and NPS samples for the detection of SARS-CoV-2 by real-time RT-PCR in patients receiving Tixagevimab/Cilgavimab, Nirmatrelvir/Ritonavir, or Sotrovimab as a treatment against SARS-CoV-2. Our results showed a good correlation between the NPS and saliva samples for each drug; moreover, comparable changes in the cycle threshold (Ct) levels in saliva and NPSs were observed both 7 days and 30 days after treatment, thus confirming that the saliva represents a good matrix for in vivo follow-up studies verifying the effectiveness of treatments against SARS-CoV-2.

ct2vl: A Robust Public Resource for Converting SARS-CoV-2 Ct Values to Viral Loads.

The amount of SARS-CoV-2 in a sample is often measured using Ct values. However, the same Ct value may correspond to different viral loads on different platforms and assays, making them difficult to compare from study to study. To address this problem, we developed ct2vl, a Python package that converts Ct values to viral loads for any RT-qPCR assay/platform. The method is novel in that it is based on determining the maximum PCR replication efficiency, as opposed to fitting a sigmoid (S-shaped) curve relating signal to cycle number. We calibrated ct2vl on two FDA-approved platforms and validated its performance using reference-standard material, including sensitivity analysis. We found that ct2vl-predicted viral loads were highly accurate across five orders of magnitude, with 1.6-fold median error (for comparison, viral loads in clinical samples vary over 10 orders of magnitude). The package has 100% test coverage. We describe installation and usage both from the Unix command-line and from interactive Python environments. ct2vl is freely available via the Python Package Index (PyPI). It facilitates conversion of Ct values to viral loads for clinical investigators, basic researchers, and test developers for any RT-qPCR platform. It thus facilitates comparison among the many quantitative studies of SARS-CoV-2 by helping render observations in a natural, universal unit of measure.

Recapitulating COVID-19 detection methods: RT-PCR, sniffer dogs and electronic nose.

In December 2019, a number of subjects presenting with an unexplained pneumonia-like illness were suspected to have a link to a seafood market in Wuhan, China. Subsequently, this illness was identified as the 2019-novel coronavirus (2019-nCoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the World Committee on Virus Classification. Since its initial identification, the virus has rapidly sperad across the globe, posing an extraordinary challenge for the medical community. Currently, the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is considered the most reliable method for diagnosing SARS-CoV-2. This procedure involves collecting oro-pharyngeal or nasopharyngeal swabs from individuals. Nevertheless, for the early detection of low viral loads, a more sensitive technique, such as droplet digital PCR (ddPCR), has been suggested. Despite the high effectiveness of RT-PCR, there is increasing interest in utilizing highly trained dogs and electronic noses (eNoses) as alternative methods for screening asymptomatic individuals for SARS-CoV-2. These dogs and eNoses have demonstrated high sensitivity and can detect volatile organic compounds (VOCs), enabling them to distinguish between COVID-19 positive and negative individuals. This manuscript recapitulates the potential, advantages, and limitations of employing trained dogs and eNoses for the screening and control of SARS-CoV-2.

[Evaluation of Viral Subgenomic RNAs and Antigen Presence in SARS-CoV-2 PCR Positive Cases]. / SARS-CoV-2 PCR Pozitif Olgularda Viral Subgenomik RNA'larin ve Antijen Varliginin Degerlendirilmesi.

Polymerase chain reaction (PCR) and antigen test (AgT) are frequently used in the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Routine PCR tests that detect the virus genome cannot determine whether the virus is infectious or not. However, detection of subgenomic RNA (sgRNA) produced during the replication period may indicate active viral infection. Active virus detection can offer various health and economic benefits from isolation time to treatment. Antigen tests are also considered as indicators of infectiousness since they can detect viruses above a certain load amount. The aim of this study was to use two different subgenomic RNAs and antigen test instead of genomic RNA to examine the relationship with each other and the clinic in terms of infectiousness. Evaluating the antigen test together with subgenomic RNA as an indicator of infectiousness may show the importance of this test. SARS-CoV-2 PCR positive 109 naso/oropharyngeal swab samples stored at -80 °C were included in the study. In order to confirm the PCR positivity of these samples, E gene PCR was performed and AgT, and E and N sgRNA quantitative real-time reverse transcription-PCR (RT-qPCR) detection was performed. Of the 109 SARSCoV-2 PCR positive samples, 83 (76.14%) had antigen test positivity, 88 (80.73%) had E gene sgRNA, 96 (88.07%) had N gene sgRNA and 97 (89%) had at least one sgRNA positivity.The antigen test was found positive in 77.3% of the samples in which at least one sgRNA was detected and in 66.7% of the negative samples and this difference was not statistically significant (p= 0.475). The difference between E sgRNA and AgT positivity was significant (p= 0.023). N sgRNA was positive in 98.9% of E sgRNA positive samples and 42.9% of the negative samples and this difference was statistically significant (p= 0.0001). The AgT positivity rate was found to be 98.15% (53/54) for cycle threshold (Ct) value ≤ 25, 57.14% (12/21) for Ct 25-30, and 52.94% (18/34) for Ct ≥ 30. The difference in antigen test positivity between E gRNA Ct value ≤ 25 and > 25, ≤ 29 and > 29, < 30 and ≥ 30 was statistically significant (p= 0.0001). Antigen test positivity appears to be associated with viral load and infectivity, as expected. In our study, it has been shown that sgRNAs and AgT which are indicators of infectiousness can be detected at least 10 days after the symptom period. Using these two tests together could detect infective individuals with higher accuracy and shorten the duration of hospital stay and isolation.

Enhanced Point-of-Care SARS-CoV-2 Detection: Integrating RT-LAMP with Microscanning.

The COVID-19 pandemic has highlighted the urgent need for rapid and accurate diagnostic methods for various infectious diseases, including SARS-CoV-2. Traditional RT-PCR methods, while highly sensitive and specific, require complex equipment and skilled personnel. In response, we developed an integrated RT-LAMP-MS assay, which combines rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) with microscanning (MS) technology for detecting SARS-CoV-2. The assay uses magnesium pyrophosphate formed during LAMP amplification as a visual marker, allowing direct observation via microscopy without the need for additional chemical indicators or probes. For the SARS-CoV-2/IC RT-LAMP-MS assay, the sample-LAMP reagent mixture was added to a microchip with SARS-CoV-2 primers and internal controls, then incubated at 62 °C for 30 min in a heat block, followed by amplification analysis using a microscanner. In clinical tests, the RT-LAMP-MS assay showed 99% sensitivity and 100% specificity, which is identical to the RT-LAMP results and comparable to the commercial AllplexTM SARS-CoV-2 assay results. Additionally, the limit of detection (LOD) was determined to be 10-1 PFU mL-1 (dynamic range: 103~10-1 PFU mL-1). The assay delivers results in 30 min, uses low-cost equipment, and demonstrates 100% reproducibility in repeated tests, making it suitable for point-of-care use in resource-limited settings.

External Quality Assessment Program for SARS-COV-2 Molecular Detection in Pakistan.

INTRODUCTION: Amid coronavirus disease 2019 (COVID-19) pandemic, accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for diagnosis management and breaking down transmission chains. We designed a national external quality assessment panel (EQAP) for SARS-CoV-2 molecular detection comprising working laboratories nationwide. METHODS: A molecular diagnostic EQA panel that consists of five samples for SARS CoV-2 testing was distributed to 141 public and private sector laboratories across country. These samples contain different concentrations of SARS-CoV-2 to evaluate the sensitivity of commercial kits available. RESULTS: Sensitivity among public and private sector laboratories was variable, particularly lower SARS-CoV-2 concentrations significantly increased the risk of false-negative tests, whereas Ct values of accurately tested SARS-CoV-2 specimens increased as concentration decreased. These findings highlighted that performance of used commercial kits was not significantly correlated to various extraction or PCR methods. CONCLUSION: This study highlights the need for a national external quality assessment panel (EQAP) in the country to improve the quality of the healthcare system while ensuring the accuracy and reliability of results. Furthermore, EQAPs can help laboratories meet accreditation and regulatory requirements. However, continued participation in EQAP is recommended for quality enhancement of laboratories.

Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.

The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

Minute level ultra-rapid and thousand copies level high-sensitive pathogen nucleic acid identification based on contactless impedance detection microsensor.

Early screening for pathogens is crucial during pandemic outbreaks. Nucleic acid testing (NAT) is a valuable method for keeping pathogens from spreading. However, the long detection time and large size of the instruments involved significantly limited the efficiency of detection. This work described an integrated NAT microsensor that facilitated rapid and extremely sensitive detection based on nucleic acid amplification (NAA) on a chip. The biochip consisted of two layers incorporating a heater, a thermometer, an interdigital electrode (IDE) and a reaction chamber. The Pt electrode based heater and thermometer were utilized to maintain a specific temperature for the sample in the chamber. The thermometer exhibited a good linear correlation with a sensitivity of 9.36 Ω/°C and the heater achieved a heating efficiency of approximately 6.5 °C/s. Multiple ions were released during NAA, resulting in a decrease in the impedance of the amplification system solution. A large signal of impedance was generated by the released ions due to its linear correlation with the logarithm of the ion concentration. With this detection principle, IDE was employed for real-time monitoring of the in-chip reaction system impedance and NAA process. Specific nucleic acids from two pathogens (SARS-CoV-2, Vibrio vulnificus) were detected with this microsensor. The samples were qualitatively analyzed on microchip within 3 min, with a limit of detection (LOD) of 103 copies/µL. The proposed sensor presented several advantages, including reduced NAT time and increased sensitivity. Consequently, it has shown significant potential in rapid and high-quality nucleic acid testing for the field of epidemic prevention.

Test accuracy of rapid diagnostic tests and reverse-transcription polymerase chain reaction against virus isolation in cell culture for assessing SARS-CoV-2 infectivity: Systematic review and meta-analysis.

We aimed to assess the performance of Ag-RDT and RT-qPCR with regard to detecting infectious SARS-CoV-2 in cell cultures, as their diagnostic test accuracy (DTA) compared to virus isolation remains largely unknown. We searched three databases up to 15 December 2021 for DTA studies. The bivariate model was used to synthesise the estimates. Risk of bias was assessed using QUADAS-2/C. Twenty studies (2605 respiratory samples) using cell culture and at least one molecular test were identified. All studies were at high or unclear risk of bias in at least one domain. Three comparative DTA studies reported results on Ag-RDT and RT-qPCR against cell culture. Two studies evaluated RT-qPCR against cell culture only. Fifteen studies evaluated Ag-RDT against cell culture as reference standard in RT-qPCR-positive samples. For Ag-RDT, summary sensitivity was 93% (95% CI 78; 98%) and specificity 87% (95% CI 70; 95%). For RT-qPCR, summary sensitivity (continuity-corrected) was 98% (95% CI 95; 99%) and specificity 45% (95% CI 28; 63%). In studies relying on RT-qPCR-positive subsamples (n = 15), the summary sensitivity of Ag-RDT was 93% (95% CI 92; 93%) and specificity 63% (95% CI 63; 63%). Ag-RDT show moderately high sensitivity, detecting most but not all samples demonstrated to be infectious based on virus isolation. Although RT-qPCR exhibits high sensitivity across studies, its low specificity to indicate infectivity raises the question of its general superiority in all clinical settings. Study findings should be interpreted with caution due to the risk of bias, heterogeneity and the imperfect reference standard for infectivity.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2.

BACKGROUND: There is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals. METHODS: This study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity. RESULTS: The research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen's kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity. CONCLUSIONS: Based on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

RT-qPCR investigation of post-mortem tissues during COVID-19.

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.

Hand-held all-in-one (HAO) self-test kit for rapid and on-site detection of SARS-CoV-2 with colorimetric LAMP.

Throughout the COVID-19 pandemic, individuals potentially infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were forcibly recalled to local or central hospitals, where the diagnostic results were obtained a couple of days after the liquid biopsies were subjected to conventional polymerase chain reaction (PCR). This slow output of such a complex and time-consuming laboratory procedure hindered its widespread application. To overcome the limitations associated with such a centralized diagnostic system, we developed a hand-held and all-in-one type test kit in which the analytical results can be obtained in only 30 min. The test kit consists of three major steps for on-site SARS-CoV-2 RNA detection: 1) virus lysis by heat, 2) RNA enrichment by membrane, and 3) real-time detection by colorimetric loop-mediated isothermal amplification (c-LAMP). The proposed device operates in a sample-to-answer format, is fully automated, and reduces dependence on traditional laboratory settings, facilitating large-scale population screening.

Direct Detection of SARS-CoV-2 RNA in Saliva with Colorimetric RT-LAMP.

RT-LAMP is an effective alternative to RT-PCR-based diagnostics, offering high specificity, sensitivity, and rapid results. One notable advantage is the robustness of its enzymes, allowing for direct amplification from crude samples without the need for prior isolation of RNA. Colorimetric LAMP is particularly attractive as it eliminates the need for complex instrumentation, making it suitable for point-of-care applications. Here, we present a comprehensive step-by-step protocol for establishing an RT-LAMP-based test for direct detection of SARS-CoV-2 genomic RNA in saliva samples using different colorimetric detection methods. Importantly, this versatile test can be easily adapted to detect emerging pathogens.

Performance characteristics of "lollipop" swabs for the diagnosis of infection with SARS-CoV-2.

BACKGROUND: Common biologic samples used to diagnose COVID-19 include nasopharyngeal, nasal, or oropharyngeal swabs, and salivary samples. The performance characteristics of a sucked "lollipop" swab to detect SARS-CoV-2 virus is assessed in four small sub-studies. METHODS: In each sub-study, a flocked swab was sucked for 20 s and submitted for PCR detection of SARS-CoV-2 virus. RESULTS: Across all studies, 52 of 69 (75.4%) COVID-19 positive participants had positive "lollipop" swabs. Twelve of the 17 COVID-19 positive participants with negative "lollipop" swabs had known corresponding cycle threshold values of >37 from their nasal/nasopharyngeal swabs, an indication of low viral load at time of sampling. In a paired samples sub-study, the sensitivity and specificity of the "lollipop" swabs were 100% and 98%. CONCLUSIONS: "Lollipop" swabs performed satisfactorily especially in individuals with acute infection of COVID-19. "Lollipop" swabs are a simple method of sample collection for detecting SARS-CoV-2 virus and warrants additional consideration.

Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel for detection of pathogens in nasopharyngeal and lower respiratory tract specimens.

This study evaluates the performance of the QIAstat-Dx Respiratory SARS-CoV-2 Panel (RS2P) for the detection of respiratory pathogens. RS2P testing was performed on 440 specimens, including 82 negatives and 358 specimens positive for 1 or more targets (520 targets initially detected). Initial testing was performed on multiple platforms during routine laboratory workflow. Specimens with discordant results on RS2P were re-tested on a different platform to obtain a consensus result based on agreement of 2/3 assays. Percent positive, negative and overall agreement (PPA, PNA, POA), as well as concordance by number of targets and CT value range were calculated. RS2P produced valid results in 439 specimens, with a POA of 91.5 % based on consensus results, with 16/31 (51.6 %) discordant specimens with >1 positive target. When individual targets were examined, PPA, PNA and POA were 93.7 %, 99.9 % and 99.6 % compared to consensus results. Overall, RS2P performed well in detection of respiratory pathogens.