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

 Heungsup Sung  ;  Dongeun Yong  ;  Chang-Seok Ki  ;  Jae-Seok Kim  ;  Moon-Woo Seong  ;  Hyukmin Lee  ;  Mi-Na Kim 
 ANNALS OF LABORATORY MEDICINE, Vol.36(5) : 457-462, 2016 
Journal Title
Issue Date
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 ; Sputum/virology*
Comparison ; DNase ; Homogenization ; MERS coronavirus ; Nucleic acid extraction ; Proteinase K ; Sputum
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 (bioM?rieux, 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.
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1. College of Medicine (의과대학) > Dept. of Laboratory Medicine (진단검사의학교실) > 1. Journal Papers
Yonsei Authors
Yong, Dong Eun(용동은) ORCID logo https://orcid.org/0000-0002-1225-8477
Lee, Hyuk Min(이혁민) ORCID logo https://orcid.org/0000-0002-8523-4126
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