De la Torre David, Astolfi-Ferreira Claudete S, Chacon Ruy D, Piantino Ferreira Antonio J
Department of Pathology, School of Veterinary Medicine, University of São Paulo, São Paulo 05508-270, Brazil.
Vet Sci. 2018 Dec 29;6(1):2. doi: 10.3390/vetsci6010002.
Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R² value of 0.999 and a slope of -3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.
禽轮状病毒A（ARtV - A）是一种影响幼鸟的病毒，在全球家禽业中会引发急性腹泻并造成经济损失。用于诊断ARtV - A的技术包括电子显微镜检查、细胞培养分离、血清学检测以及分子技术，如逆转录 - 聚合酶链反应（RT - PCR）。本研究的目的是基于编码ARtV - A结构蛋白VP6的第6节段（S6）的核苷酸序列，使用SYBR Green化学方法标准化一种实时RT - 聚合酶链反应（RT - qPCR），用于从鸟类组织和固定在FTA卡上的材料中快速检测和定量ARtV - A。结果显示所提出的靶标能有效扩增，每微升cDNA的检测限（LoD）为一个拷贝基因（CG），定量限（LoQ）为每微升10个CG。使用标准曲线确定引物的效率为95.66%，R²值为0.999，斜率为 - 3.43。使用同时感染ARtV - A、鸡细小病毒、鸡星状病毒和禽肾炎病毒的样本作为阳性对照，以及传染性支气管炎病毒、传染性法氏囊病病毒、禽呼肠孤病毒的市售疫苗和健康器官作为阴性对照来确定特异性。该技术无非特异性PCR产物和二聚体，与传统RT - PCR相比，具有更高的灵敏度和特异性，且分析时间减少了50%以上。
