Ann Lab Med.  2016 Sep;36(5):457-462. 10.3343/alm.2016.36.5.457.

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

• Sung H
• Yong D
• Ki CS
• Kim JS
• Seong MW
• Lee H
• Kim MN

• KMID: 2373576
• DOI: http://doi.org/10.3343/alm.2016.36.5.457

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.

Keyword

MERS coronavirus; Sputum; Homogenization; Nucleic acid extraction; Comparison; Proteinase K; DNase

MeSH Terms

Acetylcysteine/chemistry
Citrates/chemistry
Coronavirus Infections/diagnosis
Deoxyribonuclease I/metabolism
Endopeptidase K/metabolism
Humans
Middle East Respiratory Syndrome Coronavirus/genetics/*isolation & purification
RNA, Viral/analysis/*isolation & purification/metabolism
Real-Time Polymerase Chain Reaction
Sputum/*virology
Acetylcysteine
Citrates
Deoxyribonuclease I
Endopeptidase K
RNA, Viral

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