Veterinary and Agrochemical Research Centre (VAR-CODA-CERVA), Operational Directorate of Virology, Groeselenberg 99, B-1180 Brussels, Belgium.
J Virol Methods. 2011 Dec;178(1-2):161-70. doi: 10.1016/j.jviromet.2011.09.007. Epub 2011 Sep 17.
A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas.
一种实时聚合酶链反应（PCR）检测方法，用于快速检测非洲猪瘟病毒（ASFV），同时检测猪β-肌动蛋白作为内参，该方法已由欧盟四个非洲猪瘟（ASF）国家参考实验室（包括欧盟参考实验室）开发和验证。用于 ASFV 的引物和 TaqMan（®）探针是从 p72 基因的保守区域中选择的。新实时 PCR 检测方法的检测限为 5.7-57 个 ASFV 基因组拷贝。使用不同的实时 PCR 设备，在实验室内部和实验室之间均证明了该 PCR 检测方法具有高精度、可重复性和稳健性（CV 范围为 0.7%至 5.4%）。使用多年来在欧洲、非洲和美洲不同地理位置收集的 44 个分离株进行病毒检测特异性验证，包括来自高加索地区、撒丁岛、东非和西非的最近分离株。与 OIE 规定的常规和实时 PCR 检测方法相比，新检测方法的灵敏度得到了提高，这在检测欧洲和非洲最近暴发和监测地区采集的 281 个田间样本（170 个样本）以及通过实验感染获得的样本（111 个样本）时得到了证明。在实验感染后的早期以及在低毒力菌株感染的猪亚临床感染过程中，这种情况尤为明显（从 35dpi 到 70dpi）。该检测方法的特异性也在来自无 ASF 地区的未感染猪和野猪的 150 个样本中得到了证实。在总共 431 个测试样本中，新检测方法的阳性偏差与 OIE 推荐的 PCR 和实时 PCR 方法相比达到 21%或 26%。这种经过改进和严格验证的实时 PCR 检测方法，带有内参，将为新感染地区的猪提供一种快速、灵敏和可靠的 ASFV 检测分子工具，在流行地区进行控制，并在无 ASF 地区进行监测。
