Wongboot Warawan, Okada Kazuhisa, Chantaroj Siriporn, Kamjumphol Watcharaporn, Hamada Shigeyuki
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Nonthaburi 11000, Thailand; National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi 11000, Thailand.
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Nonthaburi 11000, Thailand; Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
J Microbiol Methods. 2018 Aug;151:76-82. doi: 10.1016/j.mimet.2018.06.006. Epub 2018 Jun 19.
Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa, have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens. We first selected the 24 oligonucleotide primer sets and hydrolysis probes conjugated with the fluorescent reporter dyes FAM, NED, or ABY, along with an internal control, and the passive reference dye ROX to establish a single-plate panel assay. The 12-duplex qPCR panel showed high linearity, with R values of 0.981-1.0 and limits of detection ranging from 1 to 10 fg for bacterial DNA (1-200 cells), 10-10 copies for viral DNA/RNA, and 10 fg for parasitic DNA (equivalent to approximately 1 parasite) per reaction. The accuracy and robustness of the assay was demonstrated in experiments using clinical stool specimens. This platform is low cost and easily customizable, and can be applied to various types of qPCR instruments and experimental designs for surveillance of acute diarrhea.
急性腹泻病是全球公共卫生关注的问题，尤其是在发展中国家。人们已经认识到多种与腹泻相关的微生物种类，包括细菌、病毒和原生动物。用于检测多种致腹泻肠道微生物的简化方法可以明确病因并有助于腹泻病的诊断。在此，我们报告一种基于定量实时（q）PCR的方法，用于同时检测粪便标本中19种疑似引起腹泻的微生物的24个靶点。我们首先选择了24对寡核苷酸引物和与荧光报告染料FAM、NED或ABY结合的水解探针，以及一个内部对照和被动参比染料ROX，以建立单孔板检测方法。12重qPCR检测板显示出高线性，细菌DNA（1 - 200个细胞）的R值为0.981 - 1.0，检测限为1至10 fg，病毒DNA/RNA为10 - 10拷贝，每个反应中寄生虫DNA为10 fg（相当于约1个寄生虫）。使用临床粪便标本的实验证明了该检测方法的准确性和稳健性。该平台成本低且易于定制，可应用于各种类型的qPCR仪器和实验设计，用于急性腹泻的监测。
