Centro Nacional de Investigación Disciplinaria en Microbiología Animal (CENID-MA), INIFAP.
Campus Toluca, Universidad del Valle de México.
J Vis Exp. 2021 May 22(171). doi: 10.3791/61429.
Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon's horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.
牛麻痹性狂犬病（Bovine paralytic rabies，BPR）是一种病毒性脑炎，在拉丁美洲具有重要的经济意义，因为它构成了重大的人畜共患病风险。在这里，我们的目的是利用实验室方案，通过定量实时逆转录聚合酶链反应（qRT-PCR）确定不同牛脑解剖结构中狂犬病病毒（Rabies virus，RABV）基因组的相对拷贝数。qRT-PCR 根据扩增后发出的荧光定量计算样品中存在的特定基因拷贝数，该荧光与样品中存在的靶核酸量成正比。与其他技术相比，这种方法具有耗时短、污染风险低以及更容易检测不同样品中的病毒核酸等优点。从六只患有狂犬病的动物的大脑中，将其分为六个解剖结构，分别为角回、小脑、大脑皮质、脑髓、脑桥和丘脑。所有大脑均基于直接免疫荧光试验被鉴定为 RABV 抗原阳性。还评估了来自四只 RABV 阴性牛的相同解剖结构的大脑。从每个结构中提取 RNA 并用于 qRT-PCR。使用体外转录核蛋白基因进行了测定 RABV 基因拷贝数的测定。用于定量病毒 RNA 的标准曲线的效率为 100%，线性度为 0.99。分析表明，根据观察到这些结构具有最高水平的 RABV，大脑皮质、脑髓和丘脑是用于 RABV 检测的理想中枢神经系统部分。该测试的特异性为 100%。所有样本均为阳性，未检测到假阳性。该方法可用于在 BPR 诊断中检测含有低水平 RABV 的样本。
