Institute of Laboratory Animals, Yamaguchi University Science Research Center, Yamaguchi, 755-8505, Japan.
Institute of Gene Research, Yamaguchi University Science Research Center, Yamaguchi, 755-8505, Japan.
BMC Microbiol. 2020 Dec 7;20(1):371. doi: 10.1186/s12866-020-02053-6.
Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques.
Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation.
The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.
四角盲囊线虫作为一种寄生性蛔虫，最常被发现在实验小鼠中，其传播途径是通过感染小鼠粪便中的卵。一种高度敏感和定量的粪便诊断工具将有助于早期发现四角盲囊线虫，以抑制感染的扩散。在这项研究中，我们使用 PCR 技术开发了一种灵敏的定量检测粪便中四角盲囊线虫的方法。
终点 PCR 显示，从感染小鼠的粪便中提取的 0.5ng 基因组 DNA 中可检测到四角盲囊线虫 DNA。为了定量检测少量的四角盲囊线虫 DNA，我们使用了锁核酸（LNA）基引物和 LNA 基 TaqMan 探针进行定量 PCR 检测（qPCR）。与使用普通寡脱氧核苷酸相比，LNA 基 DNA 的组合将检测灵敏度提高了 100 倍以上。检测到的四角盲囊线虫 DNA 的拷贝数与从感染粪便中提取的基因组 DNA 呈正相关，在基因组 DNA 20pg 至 15ng 的范围内具有简单的线性回归。为了更方便地通过粪便检测感染，我们将 LNA 基 TaqMan 检测应用于未经 DNA 纯化的粪便粗提物中。使用粪便乙醇沉淀的检测方法与直接显微镜观察的结果一致。
本研究中开发的 LNA-TaqMan 检测方法可定量检测小鼠粪便中的四角盲囊线虫感染。
