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.
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.
authors.affiliation: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea. sung@amc.seoul.kr.
authors.forename: Heungsup
authors.initials: H
authors.lastname: Sung
authors.affiliation: Department of Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Dongeun
authors.initials: D
authors.lastname: Yong
authors.affiliation: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors.forename: Chang Seok
authors.initials: CS
authors.lastname: Ki
authors.affiliation: Department of Laboratory Medicine, Hallym University College of Medicine, Hallym University Kangdong Sacred Heart Hospital, Seoul, Korea.
authors.forename: Jae Seok
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Seoul National University College of Medicine and Seoul National University Hospital, Seoul, Korea.
authors.forename: Moon Woo
authors.initials: MW
authors.lastname: Seong
authors.affiliation: Department of Laboratory Medicine, International St. Mary's Hospital, Catholic Kwandong University College of Medicine, Incheon, Korea.
authors.forename: Hyukmin
authors.initials: H
authors.lastname: Lee
authors.affiliation: Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea. mnkim@amc.seoul.kr.
authors.forename: Mi Na
authors.initials: MN
authors.lastname: Kim
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry; D002951:Citrates / Q000737:chemistry; D018352:Coronavirus Infections / Q000175:diagnosis; D003850:Deoxyribonuclease I / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D065207:Middle East Respiratory Syndrome Coronavirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D000077559:Sodium Citrate; D013183:Sputum / Q000821:virology*
chemical_list: D002951:Citrates; D012367:RNA, Viral; D000077559:Sodium Citrate; D003850:Deoxyribonuclease I; D019286:Endopeptidase K; D000111:Acetylcysteine
keywords: Comparison; DNase; Homogenization; MERS coronavirus; Nucleic acid extraction; Proteinase K; Sputum