Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays
Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays
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DOI：
10.1373/clinchem.2003.023663
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发表时间：
2004-01-01
影响因子：
9.3
通讯作者：
Peiris, JSM
中科院分区：
文献类型：
--
作者：
Poon, LLM;Chan, KH;Peiris, JSM
背景：一种新型冠状病毒（CoV）最近被确定为严重急性呼吸综合征（SARS）的病原体。我们比较了常规和实时荧光定量RT-PCR（RT-PCR）检测SARS CoV的能力，在临床标本。方法：从鼻咽抽吸物（NPA; n = 170）和粪便（n = 44）中分离的RNA样品进行逆转录和测试，我们在内部的常规RT-PCR方法。我们选择了发病后不同时间收集的98份NPA和37份粪便样本，并对SARS CoV开放阅读框（ORF）1b区进行了实时定量RT-PCR检测。比较了常规和实时定量RT-PCR检测的检出率。结果：实时荧光定量RT-PCR检测SARS冠状病毒的灵敏度高于常规RT-PCR检测方法，且与常规RT-PCR检测方法相比，实时荧光定量RT-PCR检测SARS冠状病毒的灵敏度更高。ORF 1b和N基因的实时荧光定量分析结果显示，临床标本中ORF 1b和N基因拷贝数基本一致。结论：NPA和粪便标本可用于SARS的早期诊断。建立的SARS冠状病毒实时荧光定量RT-PCR检测方法可用于SARS冠状病毒的早期检测。我们的研究结果表明，基因组RNA是临床样本中的主要病毒RNA种类。(C)2004年美国临床化学协会。
Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RTPCR) assays to detect SARS CoV in clinical specimens.Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV.Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar.Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples. (C) 2004 American Association for Clinical Chemistry.