The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100 to 10-3 ng/μL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10-5, 5 × 10-7, 2 × 10-5, and 7 × 10-7 ng/μL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.

中文翻译：

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用于鉴定野外血样中临床相关的无性物种的多重PCR检测方法。

Anaplasma（Rickettsiales：Anaplasmataceae）属，包括角cap（Anaplasma capra），牛（Anaplasma），牛（Anaplasma）vis和吞噬细胞（Anaplasma phagocytophilum），在全世界人类和兽医健康中引起多种感染。在许多情况下，已经报道了这四种无形体病原体的多重感染。我们基于针对groEL的物种特异性引物（A. capra和A. bovis），msp4（A）引入了一种新颖的多重PCR，用于同时检测A. capra，A。bovis，A。ovis和A. phagocytophilum （Ovis）和16S rRNA（A. phagocytophilum）基因。为了验证PCR反应的特异性，我们评估了四组引物来分析包含不同血液病原体的样品。多重PCR的敏感性通过扩增从感染了卡普拉曲霉，牛分枝杆菌，卵形曲霉或吞噬曲霉的绵羊血液中提取的总基因组DNA的10倍稀释液来评估。通过在三个不同的天中一式三份地测试每个反应中从感染了这些病原体的绵羊血液中提取的总基因组DNA的10倍稀释度从100到10-3 ng /μL，来评估测定的可重复性。总共175个现场血液DNA样品用于评估多重PCR与单纯PCR的可重复性。使用先前通过其他方法鉴定的血液病原体，本研究中使用的PCR引物被证实具有100％的物种特异性。具有良好重复性的多重PCR的检测下限使得能够检测卡普拉曲霉，牛曲霉，卵圆曲霉和曲霉。吞噬细胞的浓度分别为3×10-5、5×10-7、2×10-5和7×10-7 ng /μL。用于检测四种无形体种类的常规PCR和多重PCR方案之间没有显着差异（P> 0.05）。多重PCR的结果显示，卡普拉氏菌groEL基因，牛肝菌groEL基因，ovis osp msp4基因和噬菌细胞16S rRNA基因是用于临床分离物中物种鉴定的可靠靶基因，具有特异性四个目标无形体物种中的每一个。我们的研究为绵羊和山羊的无形体病原体的快速鉴别临床诊断和流行病学监测提供了有效，灵敏，特异和准确的工具。用于检测四种无形体种类的常规PCR和多重PCR方案之间没有显着差异（P> 0.05）。多重PCR的结果显示，卡普拉氏菌groEL基因，牛肝菌groEL基因，ovis osp msp4基因和噬菌细胞16S rRNA基因是用于临床分离物中物种鉴定的可靠靶基因，具有特异性四个目标无形体物种中的每一个。我们的研究为绵羊和山羊的无形体病原体的快速鉴别临床诊断和流行病学监测提供了有效，灵敏，特异和准确的工具。用于检测四种无形体种类的常规PCR和多重PCR方案之间没有显着差异（P> 0.05）。多重PCR的结果显示，卡普拉氏菌groEL基因，牛肝菌groEL基因，ovis osp msp4基因和噬菌细胞16S rRNA基因是用于临床分离物中物种鉴定的可靠靶基因，具有特异性四个目标无形体物种中的每一个。我们的研究为绵羊和山羊的无形体病原体的快速鉴别临床诊断和流行病学监测提供了有效，灵敏，特异和准确的工具。吞噬细胞16S rRNA基因是临床分离物中可用于物种鉴定的可靠靶基因，对四种靶无性种均具有特异性。我们的研究为绵羊和山羊的无形体病原体的快速鉴别临床诊断和流行病学监测提供了有效，灵敏，特异和准确的工具。吞噬细胞16S rRNA基因是临床分离物中可用于物种鉴定的可靠靶基因，对四种靶无性种均具有特异性。我们的研究为绵羊和山羊的无形体病原体的快速鉴别临床诊断和流行病学监测提供了有效，灵敏，特异和准确的工具。