Costa Alexandre D T, Jacomasso Thiago, Mattos Elaine C, Farias Aline B, Rampazzo Rita C P, Pinto Rebeka S, Tassi Walleyd, Marciano Maria Aparecida M, Pereira-Chioccola Vera Lucia, Murphy Helen R, da Silva Alexandre J, Krieger Marco A
Laboratório de Ciências e Tecnologias Aplicadas à Saúde (LaCTAS), Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz, Rua Professor Algacyr Munhoz Mader 3775, Curitiba 81350-010, Brazil.
Instituto de Biologia Molecular do Paraná (IBMP), Rua Professor Algacyr Munhoz Mader 3775, Curitiba 81350-010, Brazil.
Food Waterborne Parasitol. 2021 Jan 15;22:e00111. doi: 10.1016/j.fawpar.2021.e00111. eCollection 2021 Mar.
Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8 °C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with oocysts, as well as açai pulp and sugarcane juice tainted with trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions. (170 words).
由寄生虫引起的食源性疾病暴发长期以来一直是一个公共卫生问题。在现有的污染检测方法中，定量聚合酶链反应（qPCR）是最灵敏、最特异的方法之一。然而，如果由缺乏经验的人员使用，该方法可能会很繁琐且容易出错。此外，qPCR试剂在运输和储存时通常需要冷冻温度。我们提出了一种凝胶化反应形式，使试剂能够在2至8摄氏度下储存长达90天而不丧失性能。凝胶化过程消除了反应设置过程中大多数操作人员的失误，并使qPCR板随时可用。使用不同食品基质的人工污染样本对新的反应组成进行了灵敏度、特异性、重复性、再现性和稳定性评估。样本包括添加了卵囊的香菜叶和覆盆子果实，以及被锥鞭毛体污染的阿萨伊果肉和甘蔗汁。凝胶化qPCR和非凝胶化qPCR之间未发现显著差异。我们的结果表明，凝胶化检测可能有助于在各实验室获得更可重复的qPCR数据，从而支持监测行动。 （170字）
