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首页> 外文期刊>Journal of Clinical Microbiology >Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus
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Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus

机译：评估先进的逆转录PCR PCR方法和替代PCR靶区，用于检测重症急性呼吸综合征相关冠状病毒

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First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits—the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)—and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 × 106 and 2.8 × 106 copies/ml (sputum and endotracheal aspirates), 4.3 × 104 and 5.5 × 104 copies/ml (stool), and 5.5 × 102 and 5.2 × 102 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.
机译：严重急性呼吸系统综合症相关冠状病毒（SARS-CoV）的第一代逆转录PCR（RT-PCR）分析在相当一部分患者中产生假阴性结果。在本研究中，我们评估了两个基于复制酶（R）基因的第二代实时RT-PCR测试试剂盒-RealArt HPA冠状病毒LC试剂盒（Artus，德国汉堡）和LightCycler SARS-CoV定量试剂盒（Roche，Penzberg，德国），以及实时检测核衣壳（N）基因的RT-PCR方法。检测N基因RNA可能是有利的，因为它在细胞中含量很高。在确诊为SARS的患者的66个标本中（主要来自上下呼吸道和粪便的标本），该试剂盒的敏感性分别为70.8％（Artus）和67.1％（Roche）。 N基因测定的灵敏度为74.2％。所有敏感性的差异均无统计学意义（ P = 0.680 [方差分析]）。培养细胞最初所含的N-基因比R基因的RNA多五倍，但在病毒复制的4天中它们各自的水平会聚。在临床样本中，R和N基因RNA的中位浓度分别为1.2×10 6 和2.8×10 6 拷贝/ ml（痰和气管内抽吸物） ，4.3×10 4 和5.5×10 4 / ml（凳子），5.5×10 2 和5.2×10 2 份/样品（咽拭子和唾液）。样品类型之间的差异很大，但靶RNA类型之间没有差异。下呼吸道的所有（ n = 12）样本在所有测试中均呈阳性。总之，新颖的检测方法比第一代检测方法更加灵敏，但仍不能全面排除SARS。需要用于无感染风险的常规痰液取样方法，以改善SARS RT-PCR。

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