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

다국어 초록 (Multilingual 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.

참고문헌 (Reference)

1 Memish ZA, "Respiratory tract samples, viral load, and genome fraction yield in patients with Middle East respiratory syndrome" 210 : 1590-1594, 2014

2 Erin EM, "Optimized dialysis and protease inhibition of sputum dithiothreitol supernatants" 177 : 132-141, 2008

3 Zumla A, "Middle East respiratory syndrome" 386 : 995-1007, 2015

4 Centers for Disease Control and Prevention, "Middle East Respiratory Syndrome (MERS)"

5 Murray PR, "Microscopic and bacteriologic analysis of expectorated sputum" 50 : 339-344, 1975

6 Sweeney PJ, "Methods in molecular biology" Humana Press 305-311, 1993

7 Breitbart B, "Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing" 39 : 729-736, 2005

8 Poissy J, "Kinetics and pattern of viral excretion in biological specimens of two MERS-CoV cases" 61 : 275-278, 2014

9 Centers for Disease Control and Prevention, "Interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (PUIs) for Middle East Respiratory Syndrome Coronavirus (MERS-CoV)-Version 2.1"

10 Assiri A, "Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study" 13 : 752-761, 2013

11 Grebski E, "Effect of physical and chemical methods of homogenization on inflammatory mediators in sputum of asthma patients" 119 : 1521-1525, 2001

12 Raj VS, "Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC" 495 : 251-254, 2013

13 Wilson D, "Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens" 42 : 5913-5916, 2004

14 Kim HK, "Comparison of Anyplex II RV16 with the xTAG respiratory viral panel and Seeplex RV15 for detection of respiratory viruses" 51 : 1137-1141, 2013

15 Drosten C, "Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection" 13 : 745-751, 2013

16 Guery B, "Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission" 381 : 2265-2272, 2013

17 Kapoor M, "Clinical and laboratory findings of the first imported case of Middle East respiratory syndrome coronavirus to the United States" 59 : 1511-1518, 2014

18 Lee JH, "An appropriate lower respiratory tract sample is essential for diagnosis of Middle East Respiratory Syndrome (MERS)" 30 : 1207-1208, 2015

19 Desjardin LE, "Alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mRNA" 34 : 2435-2439, 1996