Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR
Identifieur interne : 001331 ( Main/Curation ); précédent : 001330; suivant : 001332Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR
Auteurs : Heungsup Sung [Corée du Sud] ; Dongeun Yong [Corée du Sud] ; Chang-Seok Ki [Corée du Sud] ; Jae-Seok Kim [Corée du Sud] ; Moon-Woo Seong [Corée du Sud] ; Hyukmin Lee [Corée du Sud] ; Mi-Na Kim [Corée du Sud]Source :
- Annals of Laboratory Medicine [ 2234-3806 ] ; 2016.
Descripteurs français
- KwdFr :
- ARN viral (analyse), ARN viral (isolement et purification), ARN viral (métabolisme), Acétylcystéine (), Citrates (), Coronavirus du syndrome respiratoire du Moyen-Orient (génétique), Coronavirus du syndrome respiratoire du Moyen-Orient (isolement et purification), Deoxyribonuclease I (métabolisme), Endopeptidase K (métabolisme), Expectoration (virologie), Humains, Infections à coronavirus (diagnostic), Réaction de polymérisation en chaine en temps réel.
- MESH :
- analyse : ARN viral.
- diagnostic : Infections à coronavirus.
- génétique : Coronavirus du syndrome respiratoire du Moyen-Orient.
- isolement et purification : ARN viral, Coronavirus du syndrome respiratoire du Moyen-Orient.
- métabolisme : ARN viral, Deoxyribonuclease I, Endopeptidase K.
- virologie : Expectoration.
- Acétylcystéine, Citrates, Humains, Réaction de polymérisation en chaine en temps réel.
English descriptors
- KwdEn :
- Acetylcysteine (chemistry), Citrates (chemistry), Coronavirus Infections (diagnosis), Deoxyribonuclease I (metabolism), Endopeptidase K (metabolism), Humans, Middle East Respiratory Syndrome Coronavirus (genetics), Middle East Respiratory Syndrome Coronavirus (isolation & purification), RNA, Viral (analysis), RNA, Viral (isolation & purification), RNA, Viral (metabolism), Real-Time Polymerase Chain Reaction, Sodium Citrate, Sputum (virology).
- MESH :
- chemical , analysis : RNA, Viral.
- chemical , chemistry : Acetylcysteine, Citrates.
- diagnosis : Coronavirus Infections.
- genetics : Middle East Respiratory Syndrome Coronavirus.
- isolation & purification : Middle East Respiratory Syndrome Coronavirus, RNA, Viral.
- chemical , metabolism : Deoxyribonuclease I, Endopeptidase K, RNA, Viral.
- virology : Sputum.
- Humans, Real-Time Polymerase Chain Reaction, Sodium Citrate.
Abstract
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.
We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and
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
The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.
Url:
DOI: 10.3343/alm.2016.36.5.457
PubMed: 27374711
PubMed Central: 4940489
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PMC:4940489Le document en format XML
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<author><name sortKey="Seong, Moon Woo" sort="Seong, Moon Woo" uniqKey="Seong M" first="Moon-Woo" last="Seong">Moon-Woo Seong</name>
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<author><name sortKey="Lee, Hyukmin" sort="Lee, Hyukmin" uniqKey="Lee H" first="Hyukmin" last="Lee">Hyukmin Lee</name>
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<author><name sortKey="Kim, Mi Na" sort="Kim, Mi Na" uniqKey="Kim M" first="Mi-Na" last="Kim">Mi-Na Kim</name>
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<series><title level="j">Annals of Laboratory Medicine</title>
<idno type="ISSN">2234-3806</idno>
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<imprint><date when="2016">2016</date>
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<term>Citrates (chemistry)</term>
<term>Coronavirus Infections (diagnosis)</term>
<term>Deoxyribonuclease I (metabolism)</term>
<term>Endopeptidase K (metabolism)</term>
<term>Humans</term>
<term>Middle East Respiratory Syndrome Coronavirus (genetics)</term>
<term>Middle East Respiratory Syndrome Coronavirus (isolation & purification)</term>
<term>RNA, Viral (analysis)</term>
<term>RNA, Viral (isolation & purification)</term>
<term>RNA, Viral (metabolism)</term>
<term>Real-Time Polymerase Chain Reaction</term>
<term>Sodium Citrate</term>
<term>Sputum (virology)</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>ARN viral (analyse)</term>
<term>ARN viral (isolement et purification)</term>
<term>ARN viral (métabolisme)</term>
<term>Acétylcystéine ()</term>
<term>Citrates ()</term>
<term>Coronavirus du syndrome respiratoire du Moyen-Orient (génétique)</term>
<term>Coronavirus du syndrome respiratoire du Moyen-Orient (isolement et purification)</term>
<term>Deoxyribonuclease I (métabolisme)</term>
<term>Endopeptidase K (métabolisme)</term>
<term>Expectoration (virologie)</term>
<term>Humains</term>
<term>Infections à coronavirus (diagnostic)</term>
<term>Réaction de polymérisation en chaine en temps réel</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>RNA, Viral</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Acetylcysteine</term>
<term>Citrates</term>
</keywords>
<keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ARN viral</term>
</keywords>
<keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Coronavirus Infections</term>
</keywords>
<keywords scheme="MESH" qualifier="diagnostic" xml:lang="fr"><term>Infections à coronavirus</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term>
</keywords>
<keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term>
<term>RNA, Viral</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ARN viral</term>
<term>Coronavirus du syndrome respiratoire du Moyen-Orient</term>
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<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Deoxyribonuclease I</term>
<term>Endopeptidase K</term>
<term>RNA, Viral</term>
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<term>Deoxyribonuclease I</term>
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<keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Expectoration</term>
</keywords>
<keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Sputum</term>
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<term>Real-Time Polymerase Chain Reaction</term>
<term>Sodium Citrate</term>
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<keywords scheme="MESH" xml:lang="fr"><term>Acétylcystéine</term>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>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.</p>
</sec>
<sec><title>Methods</title>
<p>We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and <italic>N</italic>
-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).</p>
</sec>
<sec><title>Results</title>
<p>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 <italic>upE</italic>
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 <italic>P</italic>
<0.0001).</p>
</sec>
<sec><title>Conclusions</title>
<p>The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.</p>
</sec>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Lee, Jh" uniqKey="Lee J">JH Lee</name>
</author>
<author><name sortKey="Lee, Cs" uniqKey="Lee C">CS Lee</name>
</author>
<author><name sortKey="Lee, Hb" uniqKey="Lee H">HB Lee</name>
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</analytic>
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<author><name sortKey="Kumar, A" uniqKey="Kumar A">A Kumar</name>
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