Becker Alice Silveira, Lopes Thaísa Regina Rocha, Pedroso Natália Hettwer, Silva Júnior José Valter Joaquim, Weiblen Rudi, Flores Eduardo Furtado
Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil; Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.
Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil; Setor de Virologia, Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Pernambuco, Brazil.
J Virol Methods. 2024 Jan;323:114853. doi: 10.1016/j.jviromet.2023.114853. Epub 2023 Nov 17.
Canine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100 % (194/194) and 99 % (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15 % (29/194) and 9 % (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.001 TCID/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32 %) positive samples selected for comparison purposes. In addition, our assays did not amplify other canine viruses associated with respiratory and neurological diseases: canine adenovirus 2, canine parainfluenza virus 2, canine herpesvirus 1 and rabies virus. Overall, we describe a high-coverage primer set for CDV detection, which represents an attractive tool for laboratory diagnosis of canine distemper.
犬瘟热病毒（CDV）对全球家养犬和野生动物构成重大威胁。分子检测是诊断该疾病最灵敏和特异的方法，然而，CDV的高遗传变异性可能会影响实验室诊断。在此，我们设计了一组用于终点（RT-PCR）和实时（RT-qPCR）检测CDV的高覆盖引物。最初，我们收集了194个完整/近乎完整的CDV基因组（GenBank），并分析了它们的高度保守区域以进行引物设计。然后，我们基于我们的引物评估了RT-PCR/RT-qPCR反应的电子覆盖范围、分析灵敏度、特异性和诊断性能。此外，将我们引物的覆盖范围及其分析灵敏度和诊断性能与用于CDV检测的常用引物组（命名为PP-I）进行了比较。我们的正向（F）和反向（R）引物与分析序列的完全匹配率分别为100%（194/194）和99%（192/194），而PP-I的F和R引物与序列的完全匹配率分别为15%（29/194）和9%（18/194）。我们的RT-PCR和RT-qPCR的检测限与PP-I引物相当（0.001 TCID/mL）。在测试的70个临床样本中，我们的RT-PCR/RT-qPCR检测有38个呈阳性，而使用PP-I引物的反应未能检测出为比较目的而选择的28个阳性样本中的9个（32%）。此外，我们的检测方法未扩增与呼吸道和神经系统疾病相关的其他犬病毒：犬腺病毒2型、犬副流感病毒2型、犬疱疹病毒1型和狂犬病病毒。总体而言，我们描述了一组用于CDV检测的高覆盖引物，这是犬瘟热实验室诊断的一个有吸引力的工具。
