Ysea Maria Alejandra Vethencourt, Umaña Mariana Cedeño, Fuentes Sofia Pereira, Campos Idalia Valerio, Carmona Misael Chinchilla
University of Medical Sciences, Laboratory of Basic Research, San José, Costa Rica.
University of Medical Sciences, Faculty of of Microbiology, San José, Costa Rica.
J Venom Anim Toxins Incl Trop Dis. 2022 May 6;28:e20210099. doi: 10.1590/1678-9199-JVATITD-2021-0099. eCollection 2022.
The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans.
DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B).
Sensitivity A was 10 fg for , 12.5 pg for or , 50 fg for spp., 225 pg for spp. and 800 fg or 8 fg for spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for 500 cysts for or , 1000 oocysts for spp. and 3600 or four vegetatives forms for PCR or nested PCR of spp., respectively.
The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as , spp., and spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.
基础寄生虫学方法固有的灵敏度限制，以及寄生虫独特的生物学特性，使得这些方法无法有效区分形态上难以辨别的物种。分子检测和鉴定技术可用于克服这些问题。本研究的目的是对文献中描述的分子聚合酶链反应（PCR）技术进行标准化，用于检测和分子鉴定人体肠道原生动物及其他病原体。
从人或动物粪便中提取DNA，粪便事先经过洗涤或在博克-德博赫拉夫改良培养基中培养。使用默克纳格尔提取试剂盒进行DNA提取。根据文献对PCR、巢式PCR或限制性片段长度多态性（RFLP）技术进行标准化。对于所进行的每项分子技术，根据所需的最低DNA量（灵敏度A）和检测到的最低生命形式数量（灵敏度B）来确定检测的灵敏度。
分别进行1780 bp PCR或310 bp巢式PCR后，溶组织内阿米巴的灵敏度A为10 fg，蓝氏贾第鞭毛虫或脆弱双核阿米巴为12.5 pg，等孢球虫属为50 fg，隐孢子虫属为225 pg，结肠小袋纤毛虫为800 fg或8 fg。溶组织内阿米巴的灵敏度B为100个包囊，蓝氏贾第鞭毛虫或脆弱双核阿米巴为500个包囊，等孢球虫属为1000个卵囊，结肠小袋纤毛虫的PCR或巢式PCR的灵敏度B分别为3600个或4个滋养体。
实现了原生动物和色素界生物的分子检测，分子鉴定使得对某些寄生虫如溶组织内阿米巴、隐孢子虫属和结肠小袋纤毛虫进行基因分型成为可能。本研究总结了用于人类和动物流行病学研究的分子技术，并有助于在肠道寄生虫构成公共卫生问题的国家调查其传播来源。
