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1、652 JID 2004:189 (15 February) Elden et al. M A J O R A R T I C L E Frequent Detection of Human Coronaviruses in Clinical Specimens from Patients with Respiratory Tract Infection by Use of a Novel Real-Time Reverse-Transcriptase Polymerase Chain Reaction Leontine J. R. van Elden,1Anton M. van Loon,1
2、Floris van Alphen,1Karin A. W. Hendriksen,1Andy I. M. Hoepelman,2 Marian G. J. van Kraaij,3Jan-Jelrik Oosterheert,2Pauline Schipper,1Rob Schuurman,1and Monique Nijhuis1 Departments of 1Virology and2Internal Medicine, Division of Acute Medicine and Infectious Diseases, University Medical Center Utrec
3、ht, Utrecht, and 3Department of Hematology, University Medical Center St. Radboud, Nijmegen, The Netherlands During the past years, human coronaviruses (HCoVs) have been increasingly identifi ed aspathogensassociated with more-severe respiratory tract infection (RTI). Diagnostic tests for HCoVs are
4、not frequently used in the routine setting. It is likely that, as a result, the precise role that HCoVs play in RTIs is greatly underestimated. We describe a rapid, sensitive, and highly specifi c quantitative real-time reverse-transcriptasepolymerasechain reaction (RT-PCR) for the detection of HCoV
5、 that can easily be implemented in the routine diagnostic setting. HCoV was detected in 28 (11%) of the 261 clinical specimens obtained from patientspresentingwithsymptoms of RTI ranging from common cold to severe pneumonia. Only 1 (0.4%) of the 243 control specimens obtained from patients without s
6、ymptoms of RTI showed the presence of HCoV. We conclude that HCoVs can be frequently detected in patients presenting with RTI. Real-time RT-PCR provides a tool for large-scale epide- miological studies to further clarify the role that coronavirus infection plays in RTI in humans. Coronaviruses are e
7、nveloped RNA viruses that can cause disease in humans and animals. The human co- ronaviruses (HCoVs) were fi rst identifi ed in 1962. They belong to the family Coronaviridae, genus coronavirus, and the 2 human strains, HCoV 229E and OC43, are divided into 2 antigenic groups. HCoVs are recognized as
8、the secondmost frequent cause of the commoncold syndrome 1. During the past years, HCoVshavemore often been believed to be responsible for severe upper and lower respiratorytract infection (RTI). They have occasionally been identifi ed as a cause of pneumonia in older adults, infants, and immunocomp
9、romised pa- tients 25. Also, in otherwise healthy adultsfor ex- ample, in military recruitsclusters of infections have Received 4 May 2003; accepted 5 August 2003; electronically published 28 January 2004. Reprints or correspondence: Dr. Leontine J.R. van Elden,Eijkman-WinklerCenter for Microbiology
10、, Infectious Diseases and Infl ammation, Dept. of Virology, Univer- sity Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands (l.vaneldenazu.nl). The Journal of Infectious Diseases2004;189:6527 ? 2004 by the Infectious Diseases Society of America. All rights reserved. 00221899/2004
11、/18903-0013$15.00 been reported as a cause of pneumonia 6. Moreover, in a recent outbreak of HCoV in Normandy, the clinical manifestation ranged from mild symptoms to pneumo- nia 7. Recently, aheightenedinterestinthecoronavirus has been documented, because a previously unknown typethatdoesnotresembl
12、etheknownHCoVsisbelieved to be responsible for the outbreaks of severe acute res- piratory syndrome (SARS) in Hong Kong and Toronto 811. These studies indicate that coronaviruses are in- creasingly identifi ed as a pathogen causing severe res- piratory illnesses and that there is a need for reliable
13、and rapid identifi cation of coronaviruses. The diagnosis of HCoV infections is hampered, in part, by thediffi cultytoreplicatein-cellcultures,whereas serologic testing is time-consuming and therefore has little clinical signifi cance. As a consequence, efforts have been made to develop more-sensiti
14、ve molecular detec- tion methods, such as reverse-transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR 12, 13. These methods have been shown to be very valuable for the detection of HCoV in different populations of patients, such as children with otitis media, patients at University of
15、 Pennsylvania Library on May 13, 2015http:/jid.oxfordjournals.org/Downloaded from Real-Time RT-PCR for Detection of HCoV JID 2004:189 (15 February) 653 Table 1.Selected primers and probes for the real-time reverse- transcriptase polymerase chain reaction of human coronavirus (HCoV)229E and HCoV-OC43
16、. HCoV type (target), primer/probeSequence, 5?r3? Nucleotide positiona 229E (N gene) N229E-1CAGTCAAATGGGCTGATGCA154173 N229E -2AAAGGGCTATAAAGAGAATAAGGTATTCT230201 N229E-pCCCTGACGACCACGTTGTGGTTCA199176 OC43 (N gene) NOC43-1CGATGAGGCTATTCCGACTAGGT577599 NOC43-2CCTTCCTGAGCCTTCAATATAGTAACC652626 NOC43-pTCCGCCTGGCACGGTACTCCCT601622 a Primer and probe positions are given according to their position on the nucleocapsid (N) gene. with multiple sclerosis, immunocompromised patients with pneumonia
