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30 September 2020 Development of a Droplet Digital PCR for Detection of Trichuriasis in Sheep
Zhichao Yu, Zhiguo Zhao, Linjun Chen, Junyan Li, Xianghong Ju
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Abstract

Trichuriasis is a serious threat to the economic development of animal husbandry. This research aimed to establish a droplet digital PCR (ddPCR) method to detect Trichuris spp. for the early diagnosis and prevention of trichuriasis in sheep. The real-time quantitative PCR (qPCR) and ddPCR methods were used for the detection of nematodes by targeted amplification of the ITS gene. Each means was evaluated to optimize the limit of detection and reproducibility. For a recombinant plasmid, the qPCR results showed that the detection limit was 31.7 copies per reaction. In contrast to qPCR, ddPCR was able to detect concentrations below 3.17 copies per reaction. Both assays exhibited good reproducibility. However, the ddPCR method was more stable for low-copy-number detection. This new assay was specific for Trichuris spp. and did not cross-react with other relevant gastrointestinal nematodes. A total of 98 clinical samples were tested with both assays. The results showed that the positive rate of ddPCR (80.6%) was higher than that of qPCR (72.4%). This method could be used as an efficient molecular biology tool to test for Trichuris spp. and could be a new valuable tool for the clinical diagnosis and prevention of trichuriasis.

© American Society of Parasitologists 2020
Zhichao Yu, Zhiguo Zhao, Linjun Chen, Junyan Li, and Xianghong Ju "Development of a Droplet Digital PCR for Detection of Trichuriasis in Sheep," Journal of Parasitology 106(5), 603-610, (30 September 2020). https://doi.org/10.1645/20-16
Published: 30 September 2020
KEYWORDS
droplet digital PCR
molecular diagnostics
real-time quantitative PCR
Sheep Feces
Trichuriasis
Trichuris
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