Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.
Ontology highlight
ABSTRACT:
SUBMITTER: Villota SD
PROVIDER: S-EPMC8459549 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundThe current gold standard in coronavirus disease (COVID-19) diagnostics is the real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal swab (NPS) samples. Alternatively, nasal swab (NS) or saliva swab (SS) specimens are used, although available data on test accuracy are limited. We examined the diagnostic accuracy of NPS/NS/SS samples for this purpose.MethodsTen patients were included after being tested positive for SARS-CoV-2 RT-PCR in NPS samples according to the National Institute of Infectious Disease guidelines. In comparison with this conventional diagnostic method, NPS/NS/SS samples were tested using the cobas 6800 systems RT-PCR device. To investigate the usefulness of the cobas method and the difference among sample types, the agreement and sensitivity were calculated. Five to six samples were collected over a total period of 5-6 d from each patient.ResultsFifty-seven sets of NPS/NS/SS samples were collected, of which 40 tested positive for COVID-19 by the conventional method. Overall, the concordance rates using the conventional method were 86.0%/70.2%/54.4% for NPS/NS/SS samples (cobas); however, for samples collected up to and including on Day 9 after disease onset (22 negative and one positive specimens), the corresponding rates were 95.7%/87.0%/65.2%. The overall sensitivity estimates were 100.0%/67.5%/37.5% for NPS/NS/SS samples (cobas). For samples up to 9 d after onset, the corresponding values were 100.0%/86.4%/63.6%.ConclusionsNS samples are more reliable than SS samples and can be an alternative to NPS samples. They can be a useful diagnostic method in the future.
| S-EPMC8006266 | biostudies-literature
Project description:IntroductionNearly all existing respiratory syncytial virus (RSV) incidence estimates are based on real-time polymerase chain reaction (RT-PCR) testing of nasal or nasopharyngeal (NP) swabs. Adding testing of additional specimen types to NP swab RT-PCR increases RSV detection. However, prior studies only made pairwise comparisons and the synergistic effect of adding multiple specimen types has not been quantified. We compared RSV diagnosis by NP swab RT-PCR alone versus NP swab plus saliva, sputum, and serology.MethodsThis was a prospective cohort study over two study periods (27 December 2021 to 1 April 2022 and 22 August 2022 to 11 November 2022) of patients aged ≥ 40 years hospitalized for acute respiratory illness (ARI) in Louisville, KY. NP swab, saliva, and sputum specimens were collected at enrollment and PCR tested (Luminex ARIES platform). Serology specimens were obtained at acute and convalescent timepoints (enrollment and 30-60-day visit). RSV detection rate was calculated for NP swab alone and for NP swab plus all other specimen type/test.ResultsAmong 1766 patients enrolled, 100% had NP swab, 99% saliva, 34% sputum, and 21% paired serology specimens. RSV was diagnosed in 56 (3.2%) patients by NP swab alone, and in 109 (6.2%) patients by NP swab plus additional specimens, corresponding to a 1.95 times higher rate [95% confidence interval (CI) 1.62, 2.34]. Limiting the comparison to the 150 subjects with all four specimen types available (i.e., NP swab, saliva, sputum, and serology), there was a 2.60-fold increase (95% CI 1.31, 5.17) compared to NP swab alone (3.3% versus 8.7%). Sensitivities by specimen type were: NP swab 51%, saliva 70%, sputum 72%, and serology 79%.ConclusionsDiagnosis of RSV in adults was several-fold greater when additional specimen types were added to NP swab, even with a relatively low percentage of subjects with sputum and serology results available. Hospitalized RSV ARI burden estimates in adults based solely on NP swab RT-PCR should be adjusted for underestimation.
| S-EPMC10163290 | biostudies-literature
Project description:ObjectivesSensitive molecular diagnostic assays are essential for COVID-19 diagnosis. We evaluated the Hecin Scientific SARS-CoV-2 nucleic acid test kit, a dual-target real-time RT-PCR assay targeting the SARS-CoV-2 N and ORF1ab genes.MethodsThe Hecin test kit's diagnostic performance in detecting SARS-CoV-2 RNA was compared to the LightMix Modular SARS and Wuhan CoV E-gene kit (TIB Molbiol) and an in-house single-tube nested real-time RT-PCR using 296 clinical specimens, 11 proficiency testing samples, and 30 low-positive deep throat saliva and nasopharyngeal swab (NPS) samples pooled into negative samples in ratios of 1:5, 1:10, and 1:30.ResultsThe limit-of-detection of the Hecin test kit was around 500 dC/mL for the N and ORF1ab targets. Sensitivity and specificity of the Hecin test kit were 98.1% (95% CI: 93.4-99.8%) and 100% (98.1-100%), respectively, when measured against the reference method. The Hecin test kit showed fair sensitivity (80%) in low-positive NPS samples pooled in ratios of 1:5 and 1:10. Its performance in pooled samples could be dramatically improved by adjusting the assay Ct cutoff.ConclusionThe Hecin test kit enables sensitive and specific detection of SARS-CoV-2 in clinical samples and pooled samples.
| S-EPMC8182820 | biostudies-literature
Project description:ObjectivesThe gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection.MethodsWe evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before the RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, c) in a RNAsnapTM buffer.ResultsAlthough with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min.ConclusionsOur findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.
| S-EPMC7833505 | biostudies-literature
Project description:We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.
| S-EPMC4390636 | biostudies-literature
Project description:Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-μL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.
| S-EPMC7939975 | biostudies-literature
Project description:SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/μl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.
| S-EPMC7775089 | biostudies-literature
Project description:BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan(R) PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 +/- 0.33 Ct values. The coefficients for inter- (1.89 +/- 0.48%) and intra-assay variation (0.85 +/- 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 +/- 0.33, without significant differences between self-designed and ABI inventoried Taqman(R) gene assays. Only two of the tested Taqman(R) ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 +/- 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 +/- 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 +/- 0.74), albeit higher inter-assay CVs (5.38 +/- 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.
| S-EPMC2262094 | biostudies-literature
Project description:BackgroundDiagnosis of COVID-19 in symptomatic patients and screening of populations for SARS-CoV-2 infection require access to straightforward, low-cost and high-throughput testing. The recommended nasopharyngeal swab tests are limited by the need of trained professionals and specific consumables and this procedure is poorly accepted as a screening method In contrast, saliva sampling can be self-administered.MethodsIn order to compare saliva and nasopharyngeal/oropharyngeal samples for the detection of SARS-CoV-2, we designed a meta-analysis searching in PubMed up to December 29th, 2020 with the key words "(SARS-CoV-2 OR COVID-19 OR COVID19) AND (salivary OR saliva OR oral fluid)) NOT (review[Publication Type]) NOT (PrePrint[Publication Type])" applying the following criteria: records published in peer reviewed scientific journals, in English, with at least 15 nasopharyngeal/orapharyngeal swabs and saliva paired samples tested by RT-PCR, studies with available raw data including numbers of positive and negative tests with the two sampling methods. For all studies, concordance and sensitivity were calculated and then pooled in a random-effects model.FindingsA total of 377 studies were retrieved, of which 50 were eligible, reporting on 16,473 pairs of nasopharyngeal/oropharyngeal and saliva samples. Meta-analysis showed high concordance, 92.5% (95%CI: 89.5-94.7), across studies and pooled sensitivities of 86.5% (95%CI: 83.4-89.1) and 92.0% (95%CI: 89.1-94.2) from saliva and nasopharyngeal/oropharyngeal swabs respectively. Heterogeneity across studies was 72.0% for saliva and 85.0% for nasopharyngeal/oropharyngeal swabs.InterpretationOur meta-analysis strongly suggests that saliva could be used for frequent testing of COVID-19 patients and "en masse" screening of populations.
| S-EPMC8191978 | biostudies-literature
Project description:Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.
| S-EPMC8662110 | biostudies-literature