设计和开发高分辨率熔解技术，通过分析 B1 和 ROP8 基因区域来分离不同类型的刚地弓形虫。

Designing and developing of high-resolution melting technique for separating different types of Toxoplasma gondii by analysis of B1 and ROP8 gene regions.

机构信息

Department of Hygiene, Science and Research Branch, Islamic Azad University, Tehran, Iran.

Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran.

出版信息

J Microbiol Methods. 2021 May;184:106188. doi: 10.1016/j.mimet.2021.106188. Epub 2021 Mar 11.

DOI:10.1016/j.mimet.2021.106188

PMID:33713726

Abstract

BACKGROUND

Determination of Toxoplasma gondii genotypes plays an important role in the health management and epidemiology of toxoplasmosis. We developed HRM analysis to differentiate genotypes of T. gondii using the B1 and ROP8 genes, through comparing the sensitivity and specificity of both genes and methods used for the detection of T. gondii.

METHODS

A total of 96 DNA samples of muscle tissue of livestock and poultry brain tissue with three standard strains RH (type I), PRU (type II) and VEG (type III) were prepared and analyzed. Three methods of nested PCR, PCR-PCR and nested-qPCR-HRM were used. Specific new primers were designed and synthesized for developing HRM. Thirty positive samples obtained from nested-qPCR-HRM were sequenced (18 B1 and 12 ROP8).

RESULTS

Overall, 87 infected samples were identified using both genes. Through the B1 gene, we could separate type I (T = 84.8 °C) from II/III types (T = 84.6 °C). Also, the ROP8 gene could separate type II (T = 84.5 °C) from I/III types (T = 84.12 °C). Highest sensitivity (100%) and specificity (78.72%) were observed by nested-qPCR-HRM assays of the B1 and ROP8 genes than by other methods, respectively. Thus, the B1 gene can be used to most accurately detect T. gondii, while the ROP8 gene was more appropriate for T. gondii genotyping. PCR-sequencing results were consistent with HRM results in most selected samples.

CONCLUSION

HRM analysis is a powerful diagnostic tool for rapid detection and determination of main clonal lineages, and even unusual T. gondii genotypes.

摘要

背景

鉴定刚地弓形虫基因型在弓形虫病的健康管理和流行病学中起着重要作用。我们使用 B1 和 ROP8 基因开发了 HRM 分析，以区分刚地弓形虫的基因型，通过比较两种基因和检测刚地弓形虫的方法的敏感性和特异性。

方法

共制备和分析了 96 份来自家畜和家禽肌肉组织以及三种标准株 RH（I 型）、PRU（II 型）和 VEG（III 型）的脑组织的 DNA 样本。使用巢式 PCR、PCR-PCR 和巢式-qPCR-HRM 三种方法。为开发 HRM 设计并合成了新的特异性引物。从嵌套-qPCR-HRM 获得的 30 个阳性样本进行测序（18 个 B1 和 12 个 ROP8）。

结果

总体而言，使用两种基因鉴定了 87 个感染样本。通过 B1 基因，我们可以将 I 型（T = 84.8°C）与 II/III 型（T = 84.6°C）分开。此外，ROP8 基因可将 II 型（T = 84.5°C）与 I/III 型（T = 84.12°C）分开。B1 和 ROP8 基因的嵌套-qPCR-HRM 检测的灵敏度（100%）和特异性（78.72%）最高，而其他方法则较低。因此，B1 基因可用于最准确地检测刚地弓形虫，而 ROP8 基因更适合刚地弓形虫的基因分型。在大多数选定的样本中，PCR 测序结果与 HRM 结果一致。