Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays.
ABSTRACT: BACKGROUND:A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. METHODS:RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. RESULTS:The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. CONCLUSIONS:NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.
Project description:We verified the analytical performance characteristics of a previously described real-time reverse transcription-PCR (RT-PCR) assay targeting the open reading frame (ORF) 1b region of the severe acute respiratory syndrome coronavirus (SARS-CoV) with RNA transcripts. We then compared it to a novel nucleocapsid gene real-time RT-PCR assay with genomic RNA. The assays differed only in the primer and probe sequences and final concentrations. A commercially available armored RNA (Ambion, Austin, Tex.) was evaluated as positive control for the ORF 1b assay. The analytical sensitivity, reproducibility, amplification efficiency, and dynamic range of the assays were similar. Both were specific for SARS-CoV as determined by testing against human CoV 229E and OC43, specimens from patients without SARS, and by BLAST searches of GenBank for primer and probe sequence homology. The armored RNA was found to be a suitable positive control for the ORF 1b assay that could be reliably recovered and amplified from a variety of clinical specimens.
Project description:The outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, was caused by a novel coronavirus (CoV), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid detection of viral nucleic acids is critical for the early identification of infected cases. We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2. The designed primers target the nucleocapsid (N) and open reading frame (ORF) 1b gene regions, where the probes discriminate SARS-CoV-2 from other human and animal CoVs. The sensitivities are one genomic copy per reaction for the N gene assay and ten copies for the ORF 1b gene assay. The overall linear detection ranges are 1-10<sup>6</sup> and 10-10<sup>6</sup> copies per reaction for the N gene assay and the ORF 1b gene assay, respectively. Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100% (23/23) and 62.5% (16/23) of clinical specimens by the N gene assay and the ORF 1b gene assay, respectively. All of the samples not detected by the ORF 1b gene assay were throat swabs, indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay. The assays developed in the present study offer alternative diagnostic tests for COVID-19.
Project description:<h4>Introduction</h4>Mounting evidence demonstrates potential for fecal-oral transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US Food and Drug Administration now requires SARS-CoV-2 testing of potential feces donors before the use of stool manufactured for fecal microbiota transplantation. We sought to develop and validate a high-sensitivity SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) procedure for testing stool specimens.<h4>Methods</h4>A modified extraction method was used with an RT-PCR assay adapted from the Centers for Disease Control and Prevention PCR protocol for respiratory specimens. Contrived specimens were created using pre-COVID-19 banked stool specimens and spiking in known concentrations of SARS-CoV-2-specific nucleic acid. The highest transcript concentration at which 2/2 or 1/2 SARS-CoV-2 targets were detected in 9/10 replicates was defined as the dual-target limit and single-target limit of detection, respectively. The clinical performance of the assay was evaluated with stool samples collected from 17 nasopharyngeal swab RT-PCR-positive patients and 14 nasopharyngeal RT-PCR-negative patients.<h4>Results</h4>The dual-target and single-target limit of detection were 56 copies/μL and 3 copies/μL, respectively. SARS-CoV-2 was detected at concentrations as low as 0.6 copies/μL. Clinical stool samples from known COVID-19-positive patients demonstrated the detection of SARS-CoV-2 in stool up to 29 days from symptom onset with a high agreement with nasopharyngeal swab tests (kappa statistic of 0.95, P value < 0.001).<h4>Discussion</h4>The described RT-PCR test is a sensitive and flexible approach for the detection of SARS-CoV-2 in stool specimens. We propose an integrated screening approach that incorporates this stool test to support continuation of fecal microbiota transplantation programs.
Project description:<h4>Background</h4> Detection of SARS-CoV-2 infections relies on the use of sensitive, accurate and high throughput RT-PCR assays. <h4>Objectives</h4> We assessed the analytical performance of the Abbott RealTime SARS-CoV-2 (RT-SARS), Alinity m SARS-CoV-2 (AlinSARS) assays and compared the clinical performance of the RT-SARS, AlinSARS, and Alinity m Resp-4-Plex (Alin4Plex) assays to the Seegene Allplex assay (Allplex) and an inhouse test (Inhouse). <h4>Results</h4> We found 100 % positive percent agreement (PPA) and 100 % negative percent agreement (NPA) comparing RT-SARS and Allplex. RT-SARS, AlinSARS and Inhouse showed 100 % NPA and 100 % PPA across all assays, except for the RdRp target of Inhouse (PPA = 84 %). Similarly, Alin4Plex and Allplex showed high agreement with specimens containing either SARS-CoV-2, influenza A, influenza B, or RSV. Detection rates of 100 % for SARS-CoV-2 at 50 copies/mL, high precision, and no cross-reactivity with non-SARS-CoV-2 respiratory pathogens were observed for RT-SARS and AlinSARS. AlinSARS detected SARS-CoV-2 in spiked throat washes and in specimens infected with SARS-CoV-2 Alpha or Beta variants. <h4>Conclusions</h4> The newly developed RT-SARS, AlinSARS, and Alin4Plex assays proved to be useful for detecting SARS-CoV-2 RNA in clinical samples.
Project description:Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS. It is believed that SARS-CoV originates from wild animals. We have developed a multitarget real-time Taqman reverse transcription-PCR (RT-PCR) assay for the quantitative detection of SARS-CoV. The sequences of the Taqman probes with a minor groove binder and the corresponding primers were based on the sequences of the N gene, open reading frame (ORF) 3, and ORF 8. The overall linear range of this assay was from at least 10(1) to 10(6) copies per reaction, and the detection limit could reach less than 10 copies per reaction. The quantification results for SARS-CoV from cell culture correlated well with those of the RT-PCR by using any two of the three sets of primer and probe used in this assay. However, the results of quantification of SARS-CoV obtained by using a few available throat swab specimens from SARS patients and the N gene as the target were almost 10 times higher than those obtained by using ORF 3 and ORF 8. Using this assay, we also detected an apparently SARS-CoV-related coronavirus in the throat swab specimens from masked palm civets in the west part of Hubei Province, People's Republic of China.
Project description:<h4>Background</h4>SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples.<h4>Results</h4>Stool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool.<h4>Conclusions</h4>Here we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.
Project description:<b>Background:</b> Faecal transplantation is an evidence-based treatment for <i>Clostridioides difficile</i>. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for faecal microbiota transplantation (FMT), spiked samples and asymptomatic inpatients in an acute surgical unit. <b>Methods:</b> Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10 <sup>-1</sup> to 10 <sup>-5</sup> and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. <b>Results:</b> In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10 <sup>-2</sup> dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10 <sup>-1</sup> with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. <b>Conclusion:</b> RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.
Project description:<h4>Introduction</h4>Smart Gene is a point-of-care (POC)-type automated molecular testing platform that can be performed with 1 min of hands-on-time. Smart Gene SARS-CoV-2 is a newly developed Smart Gene molecular assay for the detection of SARS-CoV-2. The analytical and clinical performance of Smart Gene SARS-CoV-2 has not been evaluated.<h4>Methods</h4>Nasopharyngeal and anterior nasal samples were prospectively collected from subjects referred to the local PCR center from March 25 to July 5, 2021. Two swabs were simultaneously obtained for the Smart Gene SARS-CoV-2 assay and the reference real-time RT-PCR assay, and the results of Smart Gene SARS-CoV-2 were compared to the reference real-time RT-PCR assay.<h4>Results</h4>Among a total of 1150 samples, 68 of 791 nasopharyngeal samples and 51 of 359 anterior nasal samples were positive for SARS-CoV-2 in the reference real-time RT-PCR assay. In the testing of nasopharyngeal samples, Smart Gene SARS-CoV-2 showed the total, positive and negative concordance of 99.2% (95% confidence interval [CI]: 98.4-99.7%), 94.1% (95% CI: 85.6-98.4%) and 99.7% (95% CI: 99.0-100%), respectively. For anterior nasal samples, Smart Gene SARS-CoV-2 showed the total, positive and negative concordance of 98.9% (95% CI: 97.2-99.7%), 98.0% (95% CI: 89.6-100%) and 99.0% (95% CI: 97.2-99.8%), respectively. In total, 5 samples were positive in the reference real-time RT-PCR assay and negative in the Smart Gene SARS-CoV-2 assay, whereas 5 samples were negative in the reference real-time RT-PCR assay and positive in the Smart Gene SARS-CoV-2 assay.<h4>Conclusion</h4>Smart Gene SARS-CoV-2 showed sufficient analytical performance for the detection of SARS-CoV-2 in nasopharyngeal and anterior nasal samples.
Project description:The human bocavirus (hBoV) was first described in 2005 in respiratory tract samples. The clinical relevance of hBoV is still unclear. The aim of our study was to establish a real-time PCR assay for the detection and quantification of hBoV DNA, to apply the real-time assay for the analysis of stool and serum samples for the presence of hBoV DNA, and to perform a phylogenetic analysis of the hBoV positive samples. A total of 834 nasopharyngeal aspirates (NPA), 10 serum samples, and 31 stool samples of children with acute respiratory diseases were retrospectively tested. For phylogenetic analysis, 968 bp of the VP2 gene were sequenced from 69 hBoV-positive NPA samples. The qualitative results of the real-time hBoV PCR were in good agreement with a conventional hBoV PCR. We found that 12% of the NPA were positive for hBoV DNA. The median viral load in the NPA was 4.9 x 10(3) copies/ml (range, 2.7 x 10 degrees to 1.5 x 10(11) copies/ml). There was no difference of the hBoV load in NPA between children with or without known coinfection, but the load was significantly higher in children with bronchitis than in children with the diagnosis of febrile seizures. hBoV DNA was found in 1 of 10 serum samples and in 14 of 31 stool samples. hBoV sequence identity was >99% in the VP2 region. In conclusion, hBoV DNA can be found in NPA samples at very high titers. In addition to being found in the respiratory tract, hBoV was found in stool samples. The clinical relevance of these findings remains to be determined.
Project description:<h4>Background</h4>Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool.<h4>Methods</h4>In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing.<h4>Findings</h4>The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2.<h4>Interpretation</h4>This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic.<h4>Funding</h4>National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.