Johne’s disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis. As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

中文翻译：

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一种新颖的基于实时PCR的筛查方法，用于牛粪疾病的合并粪便样品。

约翰内氏病（JD）是禽鸟分枝杆菌亚种引起的经济上重要的畜牧业传染病。副结核病。作为主要用于筛查整个牛群的血清学测试的替代方法，我们开发了一种基于ResoLight的新型实时PCR（RL-PCR）分析方法，该方法结合了粪便样本，用于检测牛群中的粪便脱落物。RL-PCR测定法包括内部扩增对照（IC），其使用与靶分子鸟分枝杆菌亚种相同的引物对进行扩增。副结核病IS 900并根据熔化温度进行区分。合并单个粪便悬浮液，并通过离心浓缩，以避免稀释效应导致灵敏度下降。与DNA提取试剂盒（Johne-PureSpin; FASMAC）结合使用时，池中最多有15个粪便样品未观察到PCR扩增的抑制作用。池大小为10时RL-PCR的检测限为10 M. avium subsp。副结核病每克粪便中的细菌数量，与单独测试的细菌数量相当。通过单独的抗体-酶联免疫吸附测定（ELISA）和使用合并的粪便进行的RL-PCR测定，筛选了12个受感染牛群中的总共2,654只动物。通过RL-PCR筛选将50只动物诊断为JD，而通过ELISA仅有5只（在RL-PCR中也呈阳性）。在7个无JD的牛群中，由于缺少IC扩增，在327个池中有4个（1.2％）的结果无效，然后分别确认动物为阴性。我们的结果表明，通过汇集的RL-PCR进行牛群筛选将促进牛群JD的监测和控制。