一种用于定量犬血液中婴儿利什曼原虫DNA的实时聚合酶链反应检测方法的开发。

The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood.

作者信息

de Paiva Cavalcanti Milena, Felinto de Brito Maria Edileuza, de Souza Wayner Vieira, de Miranda Gomes Yara, Abath Frederico G C

机构信息

Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães-FIOCRUZ, Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-420 Recife, Brazil.

出版信息

Vet J. 2009 Nov;182(2):356-8. doi: 10.1016/j.tvjl.2008.05.018. Epub 2008 Jul 7.

DOI:10.1016/j.tvjl.2008.05.018

PMID:18603455

Abstract

Recent research has demonstrated the high sensitivity of real time PCR (qPCR) in the diagnosis of Leishmania infantum infection. The goal of this study was to develop and evaluate a qPCR detection system for the diagnosis of visceral leishmaniosis (VL) in dogs. Specific primer sets were developed for the Leishmania donovani complex, in which a fragment of 132 bp of kDNA from L. infantum was amplified. The reaction was performed using the ABI PRISM 7000 system with ABI PRISM software used to carry out the analysis. When canine blood samples were assessed using this system the detection limit of the method was found to be 0.07 parasites per reaction, the efficiency was 94.17% (R(2)=0.93, slope=-3.47) and the sensitivity and specificity were 100% and 83.33% respectively. The use of such a sensitive, reproducible and rapid qPCR-based assay will be useful in the diagnosis and control of L. infantum infection in endemic areas, where serological surveys often underestimate true disease prevalence.

摘要

最近的研究表明，实时荧光定量聚合酶链反应（qPCR）在诊断婴儿利什曼原虫感染方面具有很高的灵敏度。本研究的目的是开发和评估一种用于诊断犬内脏利什曼病（VL）的qPCR检测系统。针对杜氏利什曼原虫复合体开发了特异性引物组，其中扩增了来自婴儿利什曼原虫132 bp的kDNA片段。使用ABI PRISM 7000系统进行反应，并使用ABI PRISM软件进行分析。当使用该系统评估犬血样时，发现该方法的检测限为每个反应0.07个寄生虫，效率为94.17%（R(2)=0.93，斜率=-3.47），灵敏度和特异性分别为100%和83.33%。在血清学调查往往低估真实疾病患病率的流行地区，使用这种灵敏、可重复且快速的基于qPCR的检测方法将有助于诊断和控制婴儿利什曼原虫感染。