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Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

ABSTRACT: Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the direct use of samples with an RNase inhibitor. Direct, extraction-free use of primary samples plus the RNase inhibitor produced diagnostic values of 100 % sensitivity and specificity compared to standard protocols, and these findings were validated in a second, independent laboratory.

SUBMITTER: Villota SD

PROVIDER: S-EPMC8459549 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

Villota Stephany D SD Nipaz Victoria E VE Carrazco-Montalvo Andrés A Hernandez Sarah S Waggoner Jesse J JJ Ponce Patricio P Coloma Josefina J Orlando Alberto A Cevallos Varsovia V

Journal of virological methods 20210923

Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the dir ...[more]

PMID: 34563582

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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.

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.

Project description:Respiratory tract infections are major global health issues that require rapid and accurate diagnostic methods. Multiplex quantitative PCR (qPCR) is commonly used for pathogen detection in respiratory samples. However, the optimal specimen selection for detecting bacterial pathogens is not well-explored. We evaluated the detection rates of respiratory bacteria from nasopharyngeal swabs (NPS), sputum, and combined NPS and sputum samples using multiplex qPCR (Allplex PneumoBacter Assay, Seegene). Paired NPS and sputum samples from 219 patients with acute respiratory symptoms admitted to Korea University Anam Hospital were analyzed. qPCR was performed to detect seven respiratory bacteria: Bordetella parapertussis, Bordetella pertussis, Chlamydophila pneumoniae, Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, and Streptococcus pneumoniae. Combined NPS and sputum samples (n = 92) were created from 46 pairs of positive and 46 pairs of negative signals for NPS-alone and sputum-alone samples. Sputum samples (44.3%; 97/219) had a significantly higher positivity rate compared to NPS samples (21.0%; 46/219) (P < 0.001). The 92 combined samples identified a total of 65 bacterial nucleic acids. The detection rate for the combined samples was 86.2% (56/65), which was comparable to that of sputum alone (89.2%; 58/65) and higher than that of NPS samples (50.8%; 33/65). Combining NPS and sputum samples for PCR testing may offer an alternative for bacterial pathogen detection, providing rates comparable to those of sputum alone and greater sensitivity than that of NPS alone. This combined approach could be a cost-effective method to maximize diagnostic yield, reduce the need for multiple tests, and improve the management of respiratory infections.ImportanceThis study offers important insights into refining diagnostic strategies for respiratory bacterial infections using multiplex PCR. This study finds that combining sputum and nasopharyngeal swabs into a single tube could serve as an effective alternative for detecting respiratory bacteria in adults with acute respiratory illness.

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Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

Publications

Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

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