scholarly journals CovidArray: a microarray-based assay with high sensitivity for the detection of SARS-CoV-2 in nasopharyngeal swabs

2021 ◽  
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Digital Pcr ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity

AbstractBackgroundA new coronavirus (SARS-CoV-2) caused the current Covid-19 epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false-negative are reported. Moreover, RT-qPCR requires up to 3-6 hours with most of the time involved in RNA extraction from swab samples.MethodsWe introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid phase hybridization of fluorescently labelled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 minutes. To validate the CovidArray results, we exploited the high sensitivity of the droplet digital PCR (ddPCR) technique.ResultWe correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity that matches that of the ddPCR, we were able to identify a false-negative sample.ConclusionsCovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows to reduce both the amount of false negative results and the total analysis time to about 2 hours.

scholarly journals CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs

Sensors ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 2490
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity ◽  
Negative Results

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3–6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

scholarly journals Repeat COVID-19 Molecular Testing: Correlation with Recovery of Infectious Virus, Molecular Assay Cycle Thresholds, and Analytical Sensitivity

2020 ◽  
Author(s):  
Victoria Gniazdowski ◽  
C. Paul Morris ◽  
Shirlee Wohl ◽  
Thomas Mehoke ◽  
Srividya Ramakrishnan ◽  
...  
Keyword(s):  
Infectious Virus ◽  
Clinical Symptoms ◽  
False Negative ◽  
Digital Pcr ◽  
Viral Rna ◽  
Johns Hopkins Hospital ◽  
Molecular Testing ◽  
Analytical Sensitivity ◽  
Virus Genotype ◽  
Virus Shedding

Repeat molecular testing for SARS-CoV-2 may result in scenarios including multiple positive results, positive test results after negative tests, and repeated false negative results in symptomatic individuals. Consecutively collected specimens from a retrospective cohort of COVID-19 patients at the Johns Hopkins Hospital were assessed for RNA and infectious virus shedding. Whole genome sequencing confirmed the virus genotype in patients with prolonged viral RNA shedding and droplet digital PCR (ddPCR) was used to assess the rate of false negative standard of care PCR results. Recovery of infectious virus was associated with Ct values of 18.8 ± 3.4. Prolonged viral RNA shedding was associated with recovery of infectious virus in specimens collected up to 20 days after the first positive result in patients who were symptomatic at the time of specimen collection. The use of Ct values and clinical symptoms provides a more accurate assessment of the potential for infectious virus shedding.

scholarly journals Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Paolo Poggio ◽  
Paola Songia ◽  
Chiara Vavassori ◽  
Veronica Ricci ◽  
Cristina Banfi ◽  
...  
Keyword(s):  
Present Report ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Testing ◽  
False Negative ◽  
High Sensitivity ◽  
Digital Pcr ◽  
High Rate ◽  
Viral Gene ◽  
False Negatives ◽  
Gene Copies

AbstractPatients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

scholarly journals Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6617
Author(s):  
Eva Rajh ◽  
Tina Šket ◽  
Arne Praznik ◽  
Petra Sušjan ◽  
Alenka Šmid ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Reverse Transcription ◽  
Screening Program ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Diagnostic Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Spread ◽  
One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

scholarly journals Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools

2020 ◽  
Vol 71 (16) ◽  
pp. 2073-2078 ◽  
Author(s):  
Idan Yelin ◽  
Noga Aharony ◽  
Einat Shaer Tamar ◽  
Amir Argoetti ◽  
Esther Messer ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
False Negative Rate ◽  
Positive Sample ◽  
Recent Emergence ◽  
Single Positive ◽  
Polymerase Chain ◽  
Pool Test ◽  
Current Screening

Abstract Background The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a current pandemic of unprecedented scale. Although diagnostic tests are fundamental to the ability to detect and respond, overwhelmed healthcare systems are already experiencing shortages of reagents associated with this test, calling for a lean immediately applicable protocol. Methods RNA extracts of positive samples were tested for the presence of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction, alone or in pools of different sizes (2-, 4-, 8-, 16-, 32-, and 64-sample pools) with negative samples. Transport media of additional 3 positive samples were also tested when mixed with transport media of negative samples in pools of 8. Results A single positive sample can be detected in pools of up to 32 samples, using the standard kits and protocols, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, although this may require additional amplification cycles. Single positive samples can be detected when pooling either after or prior to RNA extraction. Conclusions As it uses the standard protocols, reagents, and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for coronavirus disease 2019 would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as in close organic groups, such as hospital departments, army units, or factory shifts.

scholarly journals RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 827
Author(s):  
Paula Rodrigues de Almeida ◽  
Ana Karolina Antunes Eisen ◽  
Meriane Demoliner ◽  
Fernando Rosado Spilki
Keyword(s):  
Reverse Transcription ◽  
Tissue Concentration ◽  
Critical Role ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Assay Sensitivity ◽  
Robust Detection ◽  
Detection Techniques ◽  
Mosquito Pool

Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration.

scholarly journals Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Viral Detection ◽  
Loop Mediated Isothermal Amplification

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

scholarly journals Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR

2018 ◽  
Vol 30 (6) ◽  
pp. 807-812 ◽  
Author(s):  
Ashley C. Weiser ◽  
Korakrit Poonsuk ◽  
Sarah A. Bade ◽  
Phillip C. Gauger ◽  
Marisa Rotolo ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
False Negative ◽  
Rna Extraction ◽  
Free Water ◽  
Negative Control ◽  
Respiratory Syndrome Virus ◽  
Sample Handling ◽  
Oral Fluids

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate–phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

scholarly journals A Universal Test to Determine the Integrity of RNA, and its Suitability for Reverse Transcription, in Animal Tissue Laboratory Specimens

2007 ◽  
Vol 19 (5) ◽  
pp. 459-464
Author(s):  
H. Jane Oakey
Keyword(s):  
Quality Assurance ◽  
16S Rrna ◽  
Reverse Transcription ◽  
False Negative ◽  
Pcr Amplification ◽  
Viral Rna ◽  
Test Results ◽  
Tissue Samples ◽  
Rna Integrity ◽  
Template Material

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

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