Schares Gereon, Globokar Vrhovec Majda, Tuschy Mareen, Joeres Maike, Bärwald Andrea, Koudela Bretislav, Dubey Jitender P, Maksimov Pavlo, Conraths Franz J
National Reference Laboratory for Toxoplasmosis, Institute of Epidemiology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, 17493, Greifswald-Insel Riems, Germany.
IDEXX Laboratories, Humboldtstraße. 2, Kornwestheim, 70806, Germany.
Parasit Vectors. 2021 Jan 25;14(1):78. doi: 10.1186/s13071-020-04571-8.
Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529).
Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection.
Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection.
The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi.
The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites.
哈蒙德哈氏球虫（Hammondia hammondi）和刚地弓形虫（Toxoplasma gondii）是密切相关的原生动物寄生虫，但只有刚地弓形虫具有人畜共患性。这两种寄生虫均以猫科动物作为终末宿主，且无法通过卵囊形态进行区分。在刚地弓形虫中，一个529碱基对（bp）的重复元件（TgREP - 529）对聚合酶链反应（PCR）诊断测试具有至关重要的诊断意义。我们在哈蒙德哈氏球虫基因组中鉴定出了一个类似的重复区域（HhamREP - 529）。
基于已报道的序列，通过计算机模拟选择引物和探针，并探索最佳引物 - 探针组合，同时也纳入了先前发表的引物。使用卵囊DNA的系列稀释液测试分析灵敏度。为测试分析特异性，将从几种相关物种中分离的DNA用作对照。将新建立的TaqMan PCR（Hham - qPCR1）应用于从感染哈蒙德哈氏球虫的γ - 干扰素基因敲除（GKO）小鼠在感染后不同时间点采集的组织。
对10条正向引物和6条反向引物进行了不同组合的测试。选择了4种潜在合适的双标记探针。基于引物对（Hham275F，Hham81R）和探针（Hham222P）的一组产生了最佳结果。除了具有出色的分析特异性外，该检测方法还显示出分析灵敏度低于一个卵囊的基因组当量。对GKO小鼠组织分布的研究表明，在所有检测的器官中均存在寄生虫DNA，但程度不同，这表明在感染后42天的慢性感染状态下，各组织之间的寄生虫载量存在100至10000倍的差异。
使用哈蒙德哈氏球虫的529 - bp重复序列适用于建立定量实时PCR检测方法，因为该重复序列在单个生物体的基因组中可能存在约200次，类似于其在刚地弓形虫中的对应序列。尽管有足够的序列数据可用，但在计算机模拟中预测的引物中只有少数显示出足够的扩增；合适探针的鉴定也很困难。这与我们之前关于哈蒙德哈氏球虫529 - bp重复元件存在相当大变异的观察结果一致。
哈蒙德哈氏球虫实时PCR代表了一种重要的新型诊断工具，可用于对哈蒙德哈氏球虫及相关寄生虫进行流行病学和细胞生物学研究。
