scholarly journals A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva

2021 ◽  
Vol 13 (4) ◽  
pp. 1061-1077
Author(s):  
Jianing Yang ◽  
Mark Kidd ◽  
Alan R. Nordquist ◽  
Stanley D. Smith ◽  
Cedric Hurth ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Diagnostic Tests ◽  
Direct Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Saliva Sample ◽  
Rna Isolation ◽  
Medical Intervention ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab

Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detection-analysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARS-CoV-2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.

scholarly journals Sensitivity of Nasopharyngeal Swabs and Saliva for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Author(s):  
Alainna J Jamal ◽  
Mohammad Mozafarihashjin ◽  
Eric Coomes ◽  
Jeff Powis ◽  
Angel X Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Saliva Sample ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Sample Pair

Abstract We enrolled 91 consecutive inpatients with COVID-19 at 6 hospitals in Toronto, Canada, and tested 1 nasopharyngeal swab/saliva sample pair from each patient using real-time RT-PCR for severe acute respiratory syndrome coronavirus 2. Sensitivity was 89% for nasopharyngeal swabs and 72% for saliva (P = .02). Difference in sensitivity was greatest for sample pairs collected later in illness.

Using quantitative real-time PCR to determine the hygienic status of cattle manure

2003 ◽  
Vol 48 (4) ◽  
pp. 97-103 ◽  
Author(s):  
M. Lebuhn ◽  
M. Effenberger ◽  
A. Gronauer ◽  
P.A. Wilderer ◽  
S. Wuertz
Keyword(s):  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Cattle Manure ◽  
Detection Sensitivity ◽  
Indicator Organisms ◽  
Humic Compounds ◽  
Target Dna ◽  
High Measurement ◽  
Polymerase Chain ◽  
Well Data

We developed a suitable system of DNA extraction and real-time quantitative polymerase chain reaction (qPCR) for the specific and sensitive quantification of pathogens and other relevant (indicator) organisms in recalcitrant material such as cattle manure. PCR inhibition by coextraction of humic compounds was minimized in this system, resulting in detection sensitivity of one target DNA copy per reaction well. Data from qPCR analysis for Escherichia coli agreed with cultivation based results, but orders of magnitude more fecal enterococci, Enterobacteriaceae and Campylobacter jejuni, were determined by qPCR than by cultivation. These bacteria may have been in a potentially hazardous active but non-cultivable state. The qPCR system is much less time consuming than conventional cultivation, highly specific, can detect non-cultivable organisms, provides high measurement throughput, and is cost attractive. It should be considered as an alternative in various application areas for (prescribed routine) cultivation based assays, e.g. for biosafety and hygiene monitoring.

scholarly journals SARS-CoV-2 Detection in Gingival Crevicular Fluid

2020 ◽  
pp. 002203452097053 ◽  
Author(s):  
S. Gupta ◽  
R. Mohindra ◽  
P.K. Chauhan ◽  
V. Singla ◽  
K. Goyal ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Gingival Crevicular Fluid ◽  
Nasopharyngeal Swab ◽  
Envelope Gene ◽  
Track Record ◽  
P Gene ◽  
Elution Buffer ◽  
Novel Coronavirus ◽  
Crevicular Fluid

Understanding the pathophysiology of the coronavirus disease 2019 (COVID-19) infection remains a significant challenge of our times. The gingival crevicular fluid being representative of systemic status and having a proven track record of detecting viruses and biomarkers forms a logical basis for evaluating the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The study aimed to assess gingival crevicular fluid (GCF) for evidence of SARS-CoV-2 in 33 patients who were deemed to be COVID-19 positive upon nasopharyngeal sampling. An attempt was also made to comparatively evaluate it with saliva in terms of its sensitivity, as a diagnostic fluid for SARS-CoV-2. GCF and saliva samples were collected from 33 COVID-19–confirmed patients. Total RNA was extracted using NucliSENS easyMAG (bioMérieux) and eluted in the elution buffer. Envelope gene ( E gene) of SARS-CoV-2 and human RNase P gene as internal control were detected in GCF samples by using the TRUPCR SARS-CoV-2 RT qPCR kit V-2.0 (I) in an Applied Biosystems 7500 real-time machine. A significant majority of both asymptomatic and mildly symptomatic patients exhibited the presence of the novel coronavirus in their GCF samples. Considering the presence of SARS-CoV-2 RNA in the nasopharyngeal swab sampling as gold standard, the sensitivity of GCF and saliva, respectively, was 63.64% (confidence interval [CI], 45.1% to 79.60%) and 64.52% (CI, 45.37% to 80.77%). GCF was found to be comparable to saliva in terms of its sensitivity to detect SARS-CoV-2. Saliva samples tested positive in 3 of the 12 patients whose GCF tested negative, and likewise GCF tested positive for 2 of the 11 patients whose saliva tested negative on real-time reverse transcription polymerase chain reaction. The results establish GCF as a possible mode of transmission of SARS-CoV-2, which is the first such report in the literature, and also provide the first quantifiable evidence pointing toward a link between the COVID-19 infection and oral health.

scholarly journals Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

Plant Disease ◽  
2019 ◽  
Vol 103 (3) ◽  
pp. 475-483 ◽  
Author(s):  
Hervé Van der Heyden ◽  
Thérèse Wallon ◽  
C. André Lévesque ◽  
Odile Carisse
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Disease Incidence ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Qpcr Assay ◽  
Inoculum Concentration ◽  
Soil P ◽  
Dry Soil ◽  
Detection And Quantification

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

scholarly journals Sensitive extraction-free SARS-CoV-2 RNA virus detection using a novel RNA preparation method

2021 ◽  
Author(s):  
Bin Guan ◽  
Karen M. Frank ◽  
José O. Maldonado ◽  
Margaret Beach ◽  
Eileen Pelayo ◽  
...  
Keyword(s):  
Preparation Method ◽  
Direct Detection ◽  
Rna Virus ◽  
Rna Extraction ◽  
Virus Detection ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Reverse Transcription Pcr ◽  
Viral Transport Medium ◽  
Droplet Pcr

AbstractCurrent conventional detection of SARS-CoV-2 involves collection of a patient sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified and novel preparation method using a Chelex resin that obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital-droplet PCR was compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat-treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high throughput platforms.

scholarly journals Direct detection of Mycobacterium avium SUBSP. paratuberculosis in bovine milk by multiplex real-time PCR

2013 ◽  
Vol 29 (3) ◽  
pp. 513-525 ◽  
Author(s):  
A. Selim ◽  
M. El-Haig ◽  
E.S. Galila
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Detection Sensitivity ◽  
Mycobacterium Avium Subsp ◽  
Pcr Assay ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Pcr Targeting

This study aimed to direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng - 5 fg/?l). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriacea were tested. Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/?l and 50 fg/?l respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/?l of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/?l). To maximize the assay?s detection sensitivity, an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large scale analysis of milk samples and other clinical specimens from man and animals.

scholarly journals Comparison of saliva with healthcare workers- and patient-collected swabs in the diagnosis of COVID-19 in a large cohort

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mitnala Sasikala ◽  
Yelamanchili Sadhana ◽  
Ketavarapu Vijayasarathy ◽  
Anand Gupta ◽  
Sarala Kumari Daram ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Early Stage ◽  
Cost Effective ◽  
Hospitalized Patients ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Predictive Values ◽  
Qrt Pcr ◽  
Asymptomatic Patients ◽  
Sensitivity Specificity

Abstract Background A considerable amount of evidence demonstrates the potential of saliva in the diagnosis of COVID-19. Our aim was to determine the sensitivity of saliva versus swabs collected by healthcare workers (HCWs) and patients themselves to assess whether saliva detection can be offered as a cost-effective, risk-free method of SARS-CoV-2 detection. Methods This study was conducted in a hospital involving outpatients and hospitalized patients. A total of 3018 outpatients were tested. Of these, 200 qRT-PCR-confirmed SARS-CoV-2-positive patients were recruited for further study. In addition, 101 SARS-CoV-2-positive hospitalized patients with symptoms were also enrolled in the study. From outpatients, HCWs collected nasopharyngeal swabs (NPS), saliva were obtained. From inpatients, HCWs collected swabs, patient-collected swabs, and saliva were obtained. qRT-PCR was performed to detect SARS-CoV-2 by TAQPATH assay to determine the sensitivity of saliva detection. Sensitivity, specificity and positive/negative predictive values (PPV, NPV) of detecting SARS-CoV-2 were calculated using MedCalc. Results Of 3018 outpatients (asymptomatic: 2683, symptomatic: 335) tested by qRT-PCR, 200 were positive (males: 140, females: 60; aged 37.9 ± 12.8 years; (81 asymptomatic, 119 symptomatic). Of these, saliva was positive in 128 (64%); 39 of 81 asymptomatic (47%),89 of 119 symptomatic patients (74.8%). Sensitivity of detection was 60.9% (55.4–66.3%, CI 95%), with a negative predictive value of 36%(32.9–39.2%, CI 95%).Among 101 hospitalized patients (males:65, females: 36; aged 53.48 ± 15.6 years), with HCW collected NPS as comparator, sensitivity of saliva was 56.1% (47.5–64.5, CI 95%), specificity 63.5%(50.4–75.3, CI95%) with PPV of 77.2% and NPV of 39.6% and that of self-swab was 52.3%(44–60.5%, CI95%), specificity 56.6% (42.3–70.2%, CI95%) with PPV 77.2% and NPV29.7%. Comparison of positivity with the onset of symptoms revealed highest detection in saliva on day 3 after onset of symptoms. Additionally, only saliva was positive in 13 (12.8%) hospitalized patients. Conclusion Saliva which is easier to collect than nasopharyngeal swab is a viable alternate to detect SARS-COV-2 in symptomatic patients in the early stage of onset of symptoms. Although saliva is currently not recommended for screening asymptomatic patients, optimization of collection and uniform timing of sampling might improve the sensitivity enabling its use as a screening tool at community level.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Self-Collection of Saliva Specimens as a Suitable Alternative to Nasopharyngeal Swabs for the Diagnosis of SARS-CoV-2 by RT-qPCR

2021 ◽  
Vol 10 (2) ◽  
pp. 299
Author(s):  
Camino Trobajo-Sanmartín ◽  
Marta Adelantado ◽  
Ana Navascués ◽  
María J. Guembe ◽  
Isabel Rodrigo-Rincón ◽  
...  
Keyword(s):  
Chronic Conditions ◽  
Saliva Sample ◽  
Virus Detection ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Personal Protection ◽  
Personal Protection Equipment ◽  
Suitable Alternative ◽  
Lysis Buffer ◽  
Sensitivity Specificity

A nasopharyngeal swab is a sample used for the diagnosis of SARS-CoV-2 infection. Saliva is a sample easier to obtain and the risk of contagion for the professional is lower. This study aimed to evaluate the utility of saliva for the diagnosis of SARS-CoV-2 infection. This prospective study involved 674 patients with suspected SARS-CoV-2 infection. Paired nasopharyngeal and saliva samples were processed by RT-qPCR. Sensitivity, specificity, and kappa coefficient were used to evaluate the results from both samples. We considered the influence of age, symptoms, chronic conditions, and sample processing with lysis buffer. Of the 674 patients, 636 (94.4%) had valid results from both samples. The virus detection in saliva compared to a nasopharyngeal sample (gold standard) was 51.9% (95% CI: 46.3%–57.4%) and increased to 91.6% (95% CI: 86.7%–96.5%) when the cycle threshold (Ct) was ≤ 30. The specificity of the saliva sample was 99.1% (95% CI: 97.0%–99.8%). The concordance between samples was 75% (κ = 0.50; 95% CI: 0.45–0.56). The Ct values were significantly higher in saliva. In conclusion, saliva sample utility is limited for clinical diagnosis, but could be a useful alternative for the detection of SARS-CoV-2 in massive screening studies, when the availability of trained professionals for sampling or personal protection equipment is limited.

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