Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples
Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples
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DOI：
10.1016/j.jviromet.2014.08.008
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发表时间：
2014-11-01
影响因子：
3.1
通讯作者：
Trallero, Gloria
中科院分区：
文献类型：
--
作者：
Cabrerizo, Maria;Calvo, Cristina;Trallero, Gloria
人类肠道病毒（EV）和双埃柯病毒（HPeV）是引起新生儿和幼儿脑膜炎、脑炎和败血症样疾病等感染的重要病原体。根据EV和HPeV基因组5 ′端非编码区的保守序列设计引物和探针，建立了同时检测临床标本中EV和HPeV的实时荧光定量RT-PCR方法。使用一组25种EV和6种HPeV类型测量该技术的灵敏度、特异性和重复性。用EV引物-探针组检测到所有EV，但没有HPeV。HPeV引物-探针组仅检测到6种HPeV类型。HPeV和EV的检测下限分别为4和40 CCID 50/ml，表明该技术对这两种病毒具有高灵敏度。对53例3岁以下儿童疑似脑膜炎、败血症或发热综合征的临床样本进行了检测。其中11例（21%）检出EV，4例（7.5%）为HPeV阳性。对73%的病毒进行了分子分型。综上所述，所开发的RT-PCR方法能够有效地检测EV和HPeV，这两种病毒可以在婴儿中引起相似的临床症状。(C)2014爱思唯尔有限公司版权所有。
Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples.Primers and probe sets were designed from the conserved 5'-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers-probe set. The HPeV primers-probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40 CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs.The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses.In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants. (C) 2014 Elsevier B.V. All rights reserved.