Riahi Maliheh, Mohammadi Mohammad Ali, Afgar Ali, Kamyabi Hossein, Nasibi Saeid, Harandi Majid Fasihi
Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, 76169114115 Kerman, Iran.
Department of Medical Parasitology, School of Medicine, Kerman University of Medical Sciences, 7616914115 Kerman, Iran.
J Parasit Dis. 2020 Dec;44(4):730-736. doi: 10.1007/s12639-020-01265-x. Epub 2020 Sep 2.
Designing and implementing Cystic Echinococcosis control programs require quantitative information about the worm load and the intensity of infection in dog populations in endemic areas. So far no "probe-based" molecular quantification tool has been available for . This study was conducted in order to develop and evaluate a qPCR technique for measuring worm load of in the final host. A species-specific TaqMan probe was designed based on the available sequences in GenBank. The study was conducted in two stages. First, stool samples from an experimentally infected dog were collected in days 7, 14, 21, 28, 35 and 49, and were analyzed by real-time qPCR assay. In the second stage, 600 mg negative stool specimens were manually spiked with 1, 5, 10, 20 and 40 eggs and the specimens were analyzed using real-time qPCR. According to the standard curve analysis, 93% efficiency and coefficients of correlation (Rsq) > 0.991 were documented. Quantitative PCR assays showed an increasing signal of infection during the 7-week course of infection. As revealed by the qPCR results from week 5 onward, signals indicative of egg excretion began and reached maximum on week 7. No qPCR signal from the samples containing 1, 10 and 20 eggs was recorded, however the samples containing 5 and 40 eggs produced signals proportional to the primary DNA. The study presents a molecular tool to quantify the burden of infection in dogs. This tool could be applied for measuring the burden of infection in the definitive hosts in surveillance and control programs.
设计和实施囊性棘球蚴病控制项目需要有关流行地区犬类种群中虫负荷和感染强度的定量信息。到目前为止，还没有可用的“基于探针”的分子定量工具。本研究旨在开发和评估一种用于测量终宿主中虫负荷的qPCR技术。基于GenBank中可用的序列设计了一种物种特异性TaqMan探针。该研究分两个阶段进行。首先，在第7、14、21、28、35和49天收集实验感染犬的粪便样本，并通过实时qPCR分析。在第二阶段，将600毫克阴性粪便标本分别人工接种1、5、10、20和40个虫卵，并使用实时qPCR对标本进行分析。根据标准曲线分析，记录到93%的效率和相关系数（Rsq）>0.991。定量PCR分析显示在7周的感染过程中感染信号增加。从第5周开始的qPCR结果显示，指示虫卵排泄的信号开始出现，并在第7周达到最大值。含有1、10和20个虫卵的样本未记录到qPCR信号，然而，含有5和40个虫卵的样本产生了与原始DNA成比例的信号。该研究提出了一种分子工具来量化犬类中感染的负担。该工具可用于在监测和控制项目中测量终宿主中的感染负担。
