Decaro Nicola, Martella Vito, Ricci Dominga, Elia Gabriella, Desario Costantina, Campolo Marco, Cavaliere Nicola, Di Trani Livia, Tempesta Maria, Buonavoglia Canio
Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, Strada prov per Casamassima Km 3, 70010 Valenzano, Bari, Italy.
J Virol Methods. 2005 Dec;130(1-2):72-8. doi: 10.1016/j.jviromet.2005.06.005. Epub 2005 Jul 15.
Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.
开发了两种基因型特异性荧光逆转录聚合酶链反应(RT-PCR)检测方法,用于检测和定量腹泻犬粪便中的犬冠状病毒(CCoV)I型和II型RNA。这两种荧光检测方法均显示出高特异性、高灵敏度和高重复性,能够在约八个数量级(从10¹到10⁸拷贝的标准RNA)的线性范围内对CCoV I型和II型RNA进行精确定量。与基于凝胶的基因型特异性RT-PCR检测方法相比,荧光检测方法比传统扩增方法更灵敏、更快速,通量大幅提高。然后,使用基因型特异性荧光检测方法检测和测量从自然感染或实验感染I型、II型或两种基因型的犬采集的粪便样本中的病毒载量。在从自然感染犬采集的174份样本中,77份CCoV I型呈阳性,46份CCoV II型呈阳性。发现38只犬同时自然感染了两种基因型,I型病毒RNA滴度通常高于II型。同时,实验感染的犬排出的I型RNA滴度相对于II型更高。