Chaisi Mamohale E, Baxter Janine R, Hove Paidashe, Choopa Chimvwele N, Oosthuizen Marinda C, Brayton Kelly A, Khumalo Zamantungwa T H, Mutshembele Awelani M, Mtshali Moses S, Collins Nicola E
Department of Veterinary Tropical Diseases, University of Pretoria.
Onderstepoort J Vet Res. 2017 Jan 23;84(1):e1-e9. doi: 10.4102/ojvr.v84i1.1262.
Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.
已经开发了几种基于核酸的检测方法来检测媒介和宿主中的边缘无形体和中央无形体，这使得在流行地区选择使用的方法变得困难。我们评估了反向线印迹（RLB）杂交检测法、两种巢式聚合酶链反应（nPCR）检测法和一种双重实时定量聚合酶链反应（qPCR）检测法检测南非牛（n = 66）中边缘无形体和中央无形体感染的能力。RLB检测法对边缘无形体质粒DNA的最低检测限为2500拷贝，nPCR和qPCR检测法为250拷贝；RLB、nPCR和qPCR检测法对中央无形体质粒DNA的最低检测限分别为2500、250和25拷贝。qPCR检测法检测到的边缘无形体和中央无形体阳性样本比其他检测法更多，无论是单一感染还是混合感染。尽管qPCR和nPCR检测结果对于大多数（38个）边缘无形体阳性样本是一致的，但有13个样本使用nPCR检测边缘无形体为阴性，而使用qPCR检测为阳性。为了解释这种差异，通过对选定现场样本的msp1β基因进行克隆和测序，评估了nPCR检测法的靶序列区域。结果表明，南非边缘无形体msp1β序列在内侧正向引物（AM100）区域存在序列变异，导致假阴性。我们建议在未来的研究中使用双重qPCR检测法，因为它更敏感，并且具有对两种无形体进行定量和多重检测的优点。
