Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Surgery, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
PLoS Negl Trop Dis. 2023 Oct 19;17(10):e0011715. doi: 10.1371/journal.pntd.0011715. eCollection 2023 Oct.
Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated.
To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 μl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I.
Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases.
Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.
开发更敏感的包虫病诊断方法至关重要。本研究旨在探索一种用于检测绵羊自然感染包虫病血清中细粒棘球绦虫游离 DNA（cfDNA）的敏感 PCR 检测方法。
为了从 35 只感染羊的血清中提取 cfDNA，使用改良的酚氯仿法提取了两种不同体积（0.5 和 2 ml）的血清样本。从每个提取的样本中，使用标准 PCR 和针对 NADH 脱氢酶亚单位 I 的半巢式 PCR 分别扩增了两个 DNA 体积（5 和 10 μl）。
标准 PCR 和半巢式 PCR 分别在 35 只羊中的 8 只和 12 只血清样本中检测到了棘球蚴 DNA；然而，使用 2 ml 的血清样本时，分别检测到了 24 只和 27 只羊。通过增加模板 DNA 的体积，PCR 可以检测到 35 只羊中的 29 只和 33 只。通过随机选择 PCR 扩增子并与 GenBank 数据库进行比较，对结果进行了验证。
提取 DNA 时使用更大体积的血清，PCR 时使用更大体积的 DNA 模板，并采用半巢式 PCR 方案，将 PCR 的灵敏度提高到 95%。该方法也可应用于人类包虫病的诊断。
