Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, South Africa.
Vet Parasitol. 2010 Mar 25;168(3-4):201-11. doi: 10.1016/j.vetpar.2009.11.011. Epub 2009 Nov 20.
A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported Theileria equi-specific qPCR assay and showed that it was able to detect the 12 T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either T. equi or B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating T. equi and B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected T. equi parasite DNA in 73% of the samples, but none of the samples were positive for B. caballi, although 19 T. equi-positive samples also hybridized to the Babesia genus-specific probe. This could indicate a mixed T. equi and B. caballi infection in these samples, with either the B. caballi parasitaemia at a level below the detection limit of the B. caballi RLB probe, or the occurrence of a novel Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The B. caballi TaqMan MGB qPCR assay was able to detect B. caballi parasite DNA in 78% of the samples. The T. equi-specific qPCR assay could positively detect T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of T. equi and B. caballi infections in field samples.
建立了一种使用 TaqMan 小沟结合物（MGB）探针的定量实时聚合酶链反应（qPCR）检测方法，用于检测南非马属动物中的巴贝西虫感染。使用先前发表的 9 个巴贝西虫 18S rRNA 基因 V4 高变区的序列来设计针对独特、保守区域的引物和探针。结果表明，巴贝西虫 TaqMan MGB qPCR 检测方法高效且具有特异性。检测限定义为 95%阳性样本可检测到的浓度，确定为 0.000114%寄生红细胞（PE）。我们进一步评估了先前报道的马媾疫锥虫特异性 qPCR 检测方法，并表明它能够检测到以前在南非鉴定的 12 种马媾疫锥虫 18S rRNA 序列变体。在疾病早期，这两种 qPCR 检测方法都比间接荧光抗体试验（IFAT）和反向线印迹（RLB）更敏感。随后在南非北开普省三个种马场的 41 匹马的田间样本中测试了这些检测方法。IFAT 检测到循环的马媾疫锥虫和巴贝西虫抗体，分别在 83%和 70%的样本中。RLB 检测到 73%的样本中存在马媾疫锥虫寄生虫 DNA，但没有样本对巴贝西虫呈阳性，尽管 19 个马媾疫锥虫阳性样本也与巴贝西虫属特异性探针杂交。这可能表明这些样本中存在混合的马媾疫锥虫和巴贝西虫感染，要么是巴贝西虫寄生率低于巴贝西虫 RLB 探针的检测限，要么是发生了新的巴贝西虫基因型或物种。相比之下，qPCR 检测方法与 IFAT 相关性较好。巴贝西虫 TaqMan MGB qPCR 检测方法能够在 78%的样本中检测到巴贝西虫寄生虫 DNA。马媾疫锥虫特异性 qPCR 检测方法能够在 80%的样本中阳性检测到马媾疫锥虫 DNA。这些结果表明，qPCR 检测方法比 RLB 检测方法更敏感，可用于检测田间样本中的马媾疫锥虫和巴贝西虫感染。
