Analytical Evaluation of Visby Medical RT-PCR Portable Device for Rapid Detection of SARS-CoV-2

Extended community testing constitutes one of the main strategic pillars in controlling the COVID-19 pandemic. Reverse transcription PCR (RT-PCR) targeting the SARS-CoV-2 genome on nasopharyngeal swab samples is currently the reference test. While displaying excellent analytical sensitivity and specificity, this test is costly, often requires a substantial turnaround time, and, more importantly, is subject to reagent and other material shortages. To complement this technology, rapid antigen tests have been developed and made available worldwide, allowing cheap, quick, and decentralized SARS-CoV-2 testing. The main drawback of these tests is the reduced sensitivity when RT-PCR is the gold standard. In this study, we evaluate Visby an innovative, portable, easy-to-use RT-PCR point-of-care (POC) diagnostic device. Our retrospective analysis shows that overall, compared to the Cobas 6800 RT-qPCR assay (Roche), this RT-PCR POC technology detects SARS-CoV-2 RNA with 95% sensitivity (95%CI = 86.3–99%) and 100% specificity (95% CI = 80.5–100%). For samples with cycle-threshold values below 31, we observed 100% sensitivity (95% CI = 66.4–100%). While showing an analytical sensitivity slightly below that of a standard RT-qPCR system, the evaluated Visby RT-PCR POC device may prove to be an interesting diagnostic alternative in the COVID-19 pandemic, potentially combining the practical advantages of rapid antigen tests and the robust analytical performances of nucleic acid detection systems.

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Development and Evaluation of a new Swiss Made SARS-CoV-2 antigen-detecting rapid test

10.1101/2021.03.25.21252280 ◽

2021 ◽

Author(s):

Percevent J Ducrest

Keyword(s):

Low Income ◽

Limit Of Detection ◽

Point Of Care ◽

Rapid Test ◽

Negative Control ◽

Low Income Countries ◽

Nasopharyngeal Swab ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Gamma Irradiated

There is an urgent need in rapid diagnostic test (RDT) to detect antigen of SARS-CoV-2 to reduce the spread of COVID-19 outbreak. We have developed a rapid and simple point-of-care lateral flow immunoassay (LFIA) detecting nucleocapsid antigen of SARS-CoV-2 in 10 minutes. The aim of this study is to evaluate the diagnostic performance and analytical sensitivity of this RDT. RT-PCR positive nasopharyngeal swab samples (n=20) for SARS-CoV-2 and 40 negative control samples were studied. Analytical sensitivity was assessed using Gamma-irradiated SARS-CoV-2 and the limit of detection (LOD) was determined at 1.4 x 10^2 TCID50/ml. Overall, RDT diagnostic sensitivity was 90% (95% confidence interval [95%CI]: 67-98%) and specificity 98% (95% CI: 85-100%). The sensitivity was 100% (95% CI: 75-100%) when using only samples with a RT-PCR Cycle threshold lower than 30. This antigen RDT displays a high diagnostic accuracy for SARS‐CoV‐2 antigen detection in high COVID‐19 prevalence settings. Its use could be considered in the absence of routine RT-PCR facilities such in low-income countries.

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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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COVID-19 Biomarkers and Advanced Sensing Technologies for Point-of-Care (POC) Diagnosis

Bioengineering ◽

10.3390/bioengineering8070098 ◽

2021 ◽

Vol 8 (7) ◽

pp. 98

Author(s):

Ernst Emmanuel Etienne ◽

Bharath Babu Nunna ◽

Niladri Talukder ◽

Yudong Wang ◽

Eon Soo Lee

Keyword(s):

Response Times ◽

Rna Virus ◽

Point Of Care ◽

Protein Detection ◽

Turnaround Time ◽

Quantitative Detection ◽

Rt Pcr ◽

Specificity And Sensitivity ◽

Small Market ◽

Multiple Biomarkers

COVID-19, also known as SARS-CoV-2 is a novel, respiratory virus currently plaguing humanity. Genetically, at its core, it is a single-strand positive-sense RNA virus. It is a beta-type Coronavirus and is distinct in its structure and binding mechanism compared to other types of coronaviruses. Testing for the virus remains a challenge due to the small market available for at-home detection. Currently, there are three main types of tests for biomarker detection: viral, antigen and antibody. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) remains the gold standard for viral testing. However, the lack of quantitative detection and turnaround time for results are drawbacks. This manuscript focuses on recent advances in COVID-19 detection that have lower limits of detection and faster response times than RT-PCR testing. The advancements in sensing platforms have amplified the detection levels and provided real-time results for SARS-CoV-2 spike protein detection with limits as low as 1 fg/mL in the Graphene Field Effect Transistor (FET) sensor. Additionally, using multiple biomarkers, detection levels can achieve a specificity and sensitivity level comparable to that of PCR testing. Proper biomarker selection coupled with nano sensing detection platforms are key in the widespread use of Point of Care (POC) diagnosis in COVID-19 detection.

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Diagnosis of SARS-Cov-2 Infection by RT-PCR Using Specimens Other Than Naso- and Oropharyngeal Swabs: A Systematic Review and Meta-Analysis

Diagnostics ◽

10.3390/diagnostics11020363 ◽

2021 ◽

Vol 11 (2) ◽

pp. 363

Author(s):

Vânia M. Moreira ◽

Paulo Mascarenhas ◽

Vanessa Machado ◽

João Botelho ◽

José João Mendes ◽

...

Keyword(s):

Systematic Review ◽

Clinical Performance ◽

Point Of Care ◽

Meta Analysis ◽

High Sensitivity ◽

Nasopharyngeal Swab ◽

Sample Collection ◽

Rt Pcr ◽

Oropharyngeal Swab ◽

Nucleic Acid Assays

The rapid and accurate testing of SARS-CoV-2 infection is still crucial to mitigate, and eventually halt, the spread of this disease. Currently, nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the recommended standard sampling techniques, yet, these have some limitations such as the complexity of collection. Hence, several other types of specimens that are easier to obtain are being tested as alternatives to nasal/throat swabs in nucleic acid assays for SARS-CoV-2 detection. This study aims to critically appraise and compare the clinical performance of RT-PCR tests using oral saliva, deep-throat saliva/posterior oropharyngeal saliva (DTS/POS), sputum, urine, feces, and tears/conjunctival swab (CS) against standard specimens (NPS, OPS, or a combination of both). In this systematic review and meta-analysis, five databases (PubMed, Scopus, Web of Science, ClinicalTrial.gov and NIPH Clinical Trial) were searched up to the 30th of December, 2020. Case-control and cohort studies on the detection of SARS-CoV-2 were included. The methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS 2). We identified 1560 entries, 33 of which (1.1%) met all required criteria and were included for the quantitative data analysis. Saliva presented the higher accuracy, 92.1% (95% CI: 70.0–98.3), with an estimated sensitivity of 83.9% (95% CI: 77.4–88.8) and specificity of 96.4% (95% CI: 89.5–98.8). DTS/POS samples had an overall accuracy of 79.7% (95% CI: 43.3–95.3), with an estimated sensitivity of 90.1% (95% CI: 83.3–96.9) and specificity of 63.1% (95% CI: 36.8–89.3). The remaining index specimens could not be adequately assessed given the lack of studies available. Our meta-analysis shows that saliva samples from the oral region provide a high sensitivity and specificity; therefore, these appear to be the best candidates for alternative specimens to NPS/OPS in SARS-CoV-2 detection, with suitable protocols for swab-free sample collection to be determined and validated in the future. The distinction between oral and extra-oral salivary samples will be crucial, since DTS/POS samples may induce a higher rate of false positives. Urine, feces, tears/CS and sputum seem unreliable for diagnosis. Saliva testing may increase testing capacity, ultimately promoting the implementation of truly deployable COVID-19 tests, which could either work at the point-of-care (e.g. hospitals, clinics) or at outbreak control spots (e.g., schools, airports, and nursing homes).

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Verification and diagnostic evaluation of the RealStar® Middle East respiratory syndrome coronavirus (N gene) reverse transcription-PCR kit 1.0

Future Microbiology ◽

10.2217/fmb-2019-0067 ◽

2019 ◽

Vol 14 (11) ◽

pp. 941-948 ◽

Cited By ~ 5

Author(s):

Leonie-Sophie Hecht ◽

Angeles Jurado-Jimenez ◽

Markus Hess ◽

Hussein El Halas ◽

Gregor Bochenek ◽

...

Keyword(s):

Middle East ◽

Reverse Transcription ◽

Diagnostic Procedure ◽

Middle East Respiratory Syndrome ◽

Diagnostic Evaluation ◽

N Gene ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Confirmatory Assay ◽

Pcr Targeting

Aim: We report the diagnostic evaluation of a confirmatory reverse transcription-PCR (RT-PCR) kit targeting the Middle East respiratory syndrome coronavirus (MERS-CoV) N gene. Material & methods: 33 patient samples from two collections sites in Riyadh, Saudi Arabia, which were pre-characterized via real-time RT-PCR targeting MERS-CoV orf1a and upE, and were tested using the MERS-CoV N gene, as a confirmatory assay. This diagnostic procedure follows a two-step diagnostics scheme, recommended by the WHO. Results: 18/33 samples tested positive, 11/33 tested negative for MERS-CoV RNA and 2/33 showed uncertain results. Conclusion: The results suggest, that the RealStar® MERS-CoV (N gene) RT-PCR kit 1.0 can be considered a suitable and reliable confirmatory assay in combination with the RealStar MERS-CoV RT-PCR kit 1.0 according to the diagnostic scheme recommended by WHO.

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Evaluation of the PAX8/PPARG Translocation in Follicular Thyroid Cancer with a 4-Color Reverse-Transcription PCR Assay and Automated High-Resolution Fragment Analysis

Clinical Chemistry ◽

10.1373/clinchem.2009.134015 ◽

2010 ◽

Vol 56 (3) ◽

pp. 391-398 ◽

Cited By ~ 23

Author(s):

Alicia Algeciras-Schimnich ◽

Dragana Milosevic ◽

Bryan McIver ◽

Heather Flynn ◽

Honey V Reddi ◽

...

Keyword(s):

Thyroid Cancer ◽

High Resolution ◽

Reverse Transcription ◽

Thyroid Tissue ◽

Molecular Testing ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Fragment Analysis ◽

Reverse Transcription Pcr

Abstract Background: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. Methods: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. Results: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100–10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell–derived neoplasms. Conclusions: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

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Bioluminescent Enzyme Immunoassay for the Detection of Norovirus Capsid Antigen

Clinical and Vaccine Immunology ◽

10.1128/cvi.00427-12 ◽

2012 ◽

Vol 19 (12) ◽

pp. 1949-1954 ◽

Cited By ~ 16

Author(s):

Nozomi Sakamaki ◽

Yoshiyuki Ohiro ◽

Mitsuki Ito ◽

Mitsuru Makinodan ◽

Tsubasa Ohta ◽

...

Keyword(s):

Enzyme Immunoassay ◽

Fecal Sample ◽

Recombinant Virus ◽

Turnaround Time ◽

Rapid Diagnosis ◽

Analytical Sensitivity ◽

Good Reproducibility ◽

Reverse Transcription Pcr ◽

Response Range ◽

Coefficients Of Variation

ABSTRACTAn ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y= 0.66x −3.21,r= 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 105to 106copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis.

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Evaluation of Three Automated Nucleic Acid Extraction Systems for Identification of Respiratory Viruses in Clinical Specimens by Multiplex Real-Time PCR

BioMed Research International ◽

10.1155/2014/430650 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Yoonjung Kim ◽

Mi-Soon Han ◽

Juwon Kim ◽

Aerin Kwon ◽

Kyung-A Lee

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

False Negative ◽

Virus Detection ◽

Turnaround Time ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Nucleic Acid Extraction ◽

Negative Results

A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

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Identification of Encephalomyocarditis Virus in Clinical Samples by Reverse Transcription-PCR Followed by Genetic Typing Using Sequence Analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.36.12.3463-3467.1998 ◽

1998 ◽

Vol 36 (12) ◽

pp. 3463-3467 ◽

Cited By ~ 13

Author(s):

H. Vanderhallen ◽

F. Koenen

Keyword(s):

Sequence Analysis ◽

Reverse Transcription ◽

Molecular Techniques ◽

Encephalomyocarditis Virus ◽

Clinical Samples ◽

Nucleotide Sequence Analysis ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Field Samples ◽

Pcr Targeting

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3′ end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.

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A Chemical-Enhanced System for CRISPR-Based Nucleic Acid Detection

10.1101/2021.03.28.437376 ◽

2021 ◽

Author(s):

Zihan Li ◽

Wenchang Zhao ◽

Shixin Ma ◽

Zexu Li ◽

Yingjia Yao ◽

...

Keyword(s):

Nucleic Acid ◽

Detection System ◽

Point Of Care ◽

Detection Sensitivity ◽

Nucleic Acid Detection ◽

Chemical Additives ◽

Lateral Flow Strip ◽

Viral Pathogens ◽

Detection Systems ◽

System Robustness

The CRISPR-based nucleic acid detection systems such as SHERLOCK, DETECTR and HOLMES have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness towards practical application of CRISPR-based diagnostics.

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