About: Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

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About: Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR Goto Sponge NotDistinct Permalink

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Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR
Creator	Sung, Heungsup Ki, Chang-Seok Kim, Jae-Seok Kim, Mi-Na Lee, Hyukmin Seong, Moon-Woo Yong, Dongeun
Source	Medline; PMC
abstract	BACKGROUND: Real-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. METHODS: We 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). RESULTS: While 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). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.
has issue date	2016-06-24(xsd:dateTime)
bibo:doi	10.3343/alm.2016.36.5.457
bibo:pmid	27374711
has license	cc-by-nc
sha1sum (hex)	01aa247284f0baf43293d8ea335b70781365a985
schema:url	https://doi.org/10.3343/alm.2016.36.5.457
resource representing a document's title	Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR
has PubMed Central identifier	PMC4940489
has PubMed identifier	27374711
schema:publication	Ann Lab Med
resource representing a document's body	covid:01aa247284f0baf43293d8ea335b70781365a985#body_text
is schema:about of	named entity 'infection' named entity 'reverse transcription PCR' named entity 'samples' named entity 'MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS' named entity 'ISOLATING' covid:arg/01aa247284f0baf43293d8ea335b70781365a985 named entity 'REAL-TIME' named entity 'REVERSE TRANSCRIPTION PCR' named entity 'RNA' named entity 'SAMPLES' named entity 'PCR' named entity 'MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS' named entity 'SPUTUM' named entity 'CONTAINING' named entity 'HOMOGENIZATION' named entity 'USED' named entity 'SPUTUM' named entity 'VIRAL' named entity 'PRIOR TO' named entity 'BACKGROUND' named entity 'REVERSE TRANSCRIPTION PCR' named entity 'DIFFICULTY' named entity 'MUCUS' named entity 'OPTIMAL' named entity 'DETERMINED' named entity 'COMPARATIVE EVALUATION' named entity 'SAMPLES' named entity 'METHODS' named entity 'NUCLEIC ACIDS' named entity 'DIAGNOSE' named entity 'NUCLEIC ACIDS' named entity 'NUCLEIC ACID ISOLATION' named entity 'HOMOGENIZATION' named entity 'EXTRACTING' named entity 'ISOLATING' named entity 'METHODS' named entity 'INFECTION' named entity 'REAL-TIME' named entity 'nucleic acids' named entity 'prior' named entity 'Real-time' named entity 'acid' named entity 'rRT-PCR' named entity 'sputum' named entity 'RNA' named entity 'sputum' named entity 'infection' named entity 'nucleic acid' named entity 'Nucleic Acids' named entity 'PCR' named entity 'viruses' named entity 'adenovirus' named entity 'NALC' named entity 'MERS-CoV' named entity 'genomes' named entity 'sputum' named entity 'phosphate-buffered saline' named entity 'NALC' named entity 'DNA viruses' named entity 'ciliated columnar epithelial cells' named entity 'ciliated' named entity 'sputum' named entity 'PBS' named entity 'Bio-Rad' named entity 'NALC' named entity 'pathogens' named entity 'sputum' named entity 'RNA' named entity 'PBS' named entity 'viral capsid proteins' named entity 'serine protease' named entity 'bioMérieux' named entity '5.8' named entity 'rRNA' named entity '5.4'

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