■经典猪瘟病毒(CSFV)仍然是动物健康中最重要的病原体之一。病原体检测依赖于病毒RNA提取,然后进行RT-qPCR。需要新技术来改善护理点的诊断。
■开发了一种环介导等温扩增(LAMP)PCR技术,设计的引物考虑了所有报道的CSFV基因型。使用荧光和比色检测测试反应,与黄金标准技术相比。来自三种循环CSFV基因型的病毒株进行了测试,以及受感染动物的样本。还测试了其他病原体,以确定LAMP的特异性。除了实验室RNA提取方法,RNA释放的加热方法,评估了易于适应野外条件的情况。
■生成了三个引物组,其中一个表现得更好。该引物组证明能够在宽范围的扩增温度(60°C-68°C)下保持最佳性能。它还能够检测来自测试的三种基因型的CSFVRNA。该测定法在检测感染了来自两种不同基因型的田间菌株的动物样品方面非常有效,使用比色法和荧光法检测多个基质。LAMP检测对所有测试的无关病原体均为阴性,包括瘟病毒。荧光和比色LAMP的唯一可疑结果是针对新型瘟病毒,绵羊意大利瘟病毒(OVPV),已证明与多种CSFV诊断技术有交叉反应。然而,只有当病毒载量高于10000个病毒颗粒时,才能以可疑的结果检测OVPV.
■本研究的结果表明,LAMP可能是当前使用的CSFV分子诊断技术的重要补充。这种技术可以在偏远地区使用,考虑到它可以适应成功使用最少的设备和微创样品。新型和传统诊断技术的联合使用可能被证明是支持CSF控制的有用替代方法。
UNASSIGNED: Classical swine fever virus (
CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care.
UNASSIGNED: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported
CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating
CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated.
UNASSIGNED: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect
CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles.
UNASSIGNED: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for
CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.