Lucero David E, Ribera Wilma, Pizarro Juan Carlos, Plaza Carlos, Gordon Levi W, Peña Reynaldo, Morrissey Leslie A, Rizzo Donna M, Stevens Lori
Department of Biology, University of Vermont, Burlington, Vermont, United States of America; Vector-borne Diseases Section, Tennessee Department of Health, Nashville, Tennessee, United States of America.
Facultad de Bioquímica, Universidad de San Francisco Xavier de Chuquisaca, Sucre, Bolivia.
PLoS Negl Trop Dis. 2014 Dec 4;8(12):e3365. doi: 10.1371/journal.pntd.0003365. eCollection 2014 Dec.
In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.
METHODOLOGY/PRINCIPAL FINDINGS: We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors).
CONCLUSIONS/SIGNIFICANCE: We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.
在本研究中，我们比较了两种分子生物学技术的效用，即线粒体12S核糖体RNA基因克隆和基于水解探针的定量聚合酶链反应（qPCR），以确定用活饵鼠夹（也称为诺雷奥鼠夹）捕获的野生型恰加斯病昆虫媒介的血餐来源。从玻利维亚丘基萨卡省安第斯高地六个地理定位的诱捕地点收集了14只瓜亚纳锥蝽。
方法/主要发现：我们通过克隆分析检测到四种血餐来源：七个样本对人类（智人）呈阳性，五个对鸡（原鸡）、纯色黑鹂呈阳性，一个对负鼠呈阳性。使用qPCR分析，我们检测到鸡（13只媒介）、人类（14只媒介）的血餐以及另一种血餐来源，犬属（4只媒介）。
结论/意义：我们表明，12S PCR产物的克隆避免了基于先验知识设计引物所带来的偏差，检测到了以前未考虑的血餐来源，并且物种特异性qPCR更敏感。通过克隆分析鉴定为特定血餐来源呈阳性的所有样本，通过qPCR也呈阳性。然而，并非所有通过qPCR呈阳性的样本通过克隆也呈阳性。我们表明，将克隆分析与基于高度敏感水解探针的qPCR分析相结合的方法，能够更全面地了解昆虫病媒的血餐来源。
