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1、SARS Coronavirus Detection Andreas Nitsche,* Brunhilde Schweiger,* Heinz Ellerbrok,* Matthias Niedrig,* and Georg Pauli* We developed a set of three real-time reverse tran- scriptionpolymerase chain reaction (PCR) assays that amplify three different regions of the SARS-associated coronavirus (SARS-C
2、oV), can be run in parallel or in a sin- gle tube, and can detect 10 genome equivalents of SARS- CoV. The assays consider all currently available SARS-CoV sequences and are optimized for two promi- nent real-time PCR platforms. The Study Recently, a new coronavirus was identified as the suspected ca
3、usative agent of an increased number of atypical pneumonia cases reported from Hong Kong, Singapore, Vietnam, and Canada (14). Subsequent publications demonstrated that this new coronavirus was detectable in patients with severe acute respiratory syndrome (SARS) (5,6), classified according to the Wo
4、rld Health Organizations case definition (7). During the first 6 months of 2003, a total of 8,422 patients were affected. This fact, together with the reappearance of the SARS- associated coronavirus (SARS-CoV) in China in late 2003, makes it clear that rapid and reliable diagnostic tools are essent
5、ial for accurate disease reporting and subsequent disease management. Because a defined treatment program and vaccination strategy are lacking, the main strategy to counteract the spread of this emerging virus is timely identification and isolation of infected persons. SARS patientstypical initial s
6、ymptoms include fever, cough, and headache, similar to many acute viral respiratory infections. Therefore, molecular-based diagnostic methods are applied to rapidly identify SARS-CoVinfected persons. Recently, nested and real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assays to det
7、ect SARS-CoV have been published (5,8). These assaysthe first tools to detect SARS-CoV in patients with SARSwere based on the short stretches of viral sequence identified as the RNA- directed RNA polymerase of a new microbe. Subsequently, sequences from several SARS-CoV iso- lates were determined, a
8、nd all of these sequences were closely related, as would be expected during the clustered outbreaks in 2003. However, the genomes of RNAviruses, including those of coronaviruses, tend to vary over time and with location (912). Recently, the sequence variations of SARS-CoV during the first epidemic p
9、hases in China in 2003 were reported. The neutral mutation rate for SARS- CoV was almost constant and similar to that of known RNA viruses; the S protein, responsible for virus-host receptor recognition, displayed the most extensive amino acid changes (13). In addition, the sequence analysis of isol
10、ates from recent SARS patients in China in 2004 has shown that 98.8%99.4% of the 3,768 bases of S gene, 99% of 658 bases of M gene, and 99% of 1,068 bases of N gene are isogenous with those submitted to public databas- es, which date back to the first epidemic in spring 2003 (14). However, even thes
11、e minimal changes could render existing PCR assays ineffective should SARS-CoV reemerge (15). To improve the ability to detect SARS-CoV safely and reduce the risk of eliciting false-negative results caused by genome sequence variations, we established three individual real-time RT-PCR assays. Target
12、 sequences were chosen by using the following criteria: 1) the regions are distributed over the whole genome, including the nonstructural polyprotein 1a and 1ab genes and the spike glycoprotein gene (Table 1); 2) the regions are highly conserved among the 89, 90, and 100 respective sequences availab
13、le in public sequence databases; 3) the regions are suitable for the design of a real-time RT-PCR assay; and 4) the designed primers, 5-nuclease probes, and amplicons displayed no considerable homology to other viruses, including human CoV OC43 and 229E in BLAST searches (available from http:/www.nc
14、bi.nlm.nih.gov/BLAST/). These assays were based on the fluorogenic oligoprobe chemistry, which uses the 5-exonuclease activity of the DNA polymerase to generate a more specific signal than that produced by the use of SYBR Green I (8). The real- time RT-PCR assays were successfully run on the Applied
15、 Biosystems real-time PCR systems (SDS7700 and SDS7000; Applied Biosystems, Foster City, CA) as well as on the Roche LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). All assays were designed as one- step RT-PCR reactions to be run under identical conditions on the respective PCR platform. Th
16、is system allowed the simultaneous detection of different SARS-CoV regions in a single PCR run. Moreover, we could combine the three assays in a single tube, which might be important when clinical material is limited. Finally, the assays were com- pared to the 5-nuclease assay published recently (5) and to a commercially available real-time PCR kit (Real-Art HPA-Coronavirus LC RT PCR Reagents, Artus GmbH, Hamburg, Germany). After optimization of primer and 5-nuclease probe concentration and
