Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces
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METHODOLOGY ARTICLE
Open Access
Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces Keishiro Isayama1, Kenji Watanabe2, Mariko Okamoto3, Tomoaki Murata1 and Yoichi Mizukami2*
Abstract Background: 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. Results: 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. Conclusion: The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces. Keywords: Locked nucleic acid, TaqMan assay, A. tetraptera, Mice, PCR, Faeces
Background Aspiculuris tetraptera and Syphacia obvelata are parasitic pinworms that are most frequently detected in laboratory mice. A. tetraptera was found a few times in mice during routine health surveillance in our animal facilities. Infection of A. tetraptera with mice occurred at a frequency of 3–90% in most animal facilities, which may be affected by the breeding environment and detection system. The prevalence of A. tetraptera in wild mouse populations is estimated to be much higher than that in animal facilities * Correspondence: [email protected] 2 Institute of Gene Research, Yamaguchi University Science Research Center, Yamaguchi 755-8505, Japan Full list of author information is available at the end of the article
[1]. The transmission of A. tetraptera to other mice is mainly mediated by eggs [2]. The laboratory mice often ingest faeces that contain pinworm eggs, and the eggs hatch into larvae that grow into adult pinworms in the proximal colon. The pinworms in the colon spawn the eggs, which are excreted together with the mouse faeces. Infection among mice is widely transmitted by the intake of faeces, including eggs in the same cage. Detection and extermination of A. tetrapter
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