ArticleAnnals of laboratory medicine2016
Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.
Article in Annals of laboratory medicine, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers, 1 of them a synthesis that pooled it.
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Who cites it
24 citing papers in PubMed, 1 synthesis or guideline pooled it, 35 citations in OpenAlex.
- Practical Guidance for Clinical Microbiology Laboratories: Viruses Causing Acute Respiratory Tract Infections.Clinical microbiology reviews · 2019Guideline
- Comparative evaluation of proteinase K and dithiothreitol as pretreatments for extracting nucleic acids from respiratory samples for multiplex PCR.BMC microbiology · 2025Article
- Nebulization of 2% lidocaine has no detectable impact on the healthy equine respiratory microbiota.PloS one · 2025Article
- Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay.Scientific reports · 2023Article
- A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.Advanced biomedical research · 2023Article
- Comparing SARS-CoV-2 Viral Load in Human Saliva to Oropharyngeal Swabs, Nasopharyngeal Swabs, and Sputum: A Systematic Review and Meta-Analysis.The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale · 2023Review
- Identification and quantification of bioactive compounds suppressing SARS-CoV-2 signals in wastewater-based epidemiology surveillance.Water research · 2022Article
- Article
- Automated molecular testing of saliva for SARS-CoV-2 detection.Diagnostic microbiology and infectious disease · 2021Article
- Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR.PloS one · 2021Article
- Chinese Expert Consensus on the Nucleic Acid Detection of SARS-CoV-2.Annals of translational medicine · 2020Review
- COVID-19 Molecular Testing in Korea: Practical Essentials and Answers From Experts Based on Experiences of Emergency Use Authorization Assays.Annals of laboratory medicine · 2020Article
- Quality of Ribonucleic Acid Extraction for Real-Time Reverse Transcription-PCR (rRT-PCR) of SARS-CoV-2: Importance of Internal Control Monitoring.Annals of laboratory medicine · 2020Article
- Nucleic Acid and Immunological Diagnostics for SARS-CoV-2: Processes, Platforms and Pitfalls.Diagnostics (Basel, Switzerland) · 2020Review
- Coronaviruses in wastewater processes: Source, fate and potential risks.Environment international · 2020Review
- Nationwide External Quality Assessment of SARS-CoV-2 Molecular Testing, South Korea.Emerging infectious diseases · 2020Article
- Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits.Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology · 2020Article
- Expert consensus-based laboratory testing of SARS-CoV-2.Journal of thoracic disease · 2020Review
- Direct Clinical Evidence Recommending the Use of Proteinase K or Dithiothreitol to Pretreat Sputum for Detection of SARS-CoV-2.Frontiers in medicine · 2020Article
- Underlying trend, seasonality, prediction, forecasting and the contribution of risk factors: an analysis of globally reported cases of Middle East Respiratory Syndrome Coronavirus.Epidemiology and infection · 2018Article
Corrections and comments
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Authors and funding
7 authors at 7 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundReal-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum.
methodsWe evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea).
resultsWhile analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001).
conclusionsThe PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.
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