Varela-M Rubén E, Arias Jinney Stefany, Velásquez Luz Elena
Unidad de Biología Molecular y Computacional, Programa de Estudio y Control de Enfermedades Tropicales (PECET), Universidad de Antioquia, Medellín, Colombia Facultad de Ciencias Básicas, Universidad Santiago de Cali, Cali, Colombia.
Biomedica. 2018 Mar 15;38(1):111-119. doi: 10.7705/biomedica.v38i0.3407.
Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis.
To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus.
Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control.
The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%).
A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.
管圆线虫病是一种由管圆线虫引起的疾病，在全球范围内均有发生。对人类和动物健康影响最大的感染是由广州管圆线虫、哥斯达黎加管圆线虫和血管圆线虫引起的。人类疾病的临床形式为嗜酸性粒细胞性脑膜炎和腹部管圆线虫病，而对犬类最常见的影响是心肺损伤。由于这些寄生虫的中间宿主——非洲大蜗牛在全球范围内传播，该病被视为一种新兴疾病。目前针对管圆线虫属的诊断方法较少，且不具有特异性、成本高且灵敏度不高。迫切需要开发一种灵敏、特异且易于使用的诊断工具来控制人类和动物的管圆线虫病。
开发一种用于鉴定三种致病性管圆线虫的多重定量聚合酶链反应（qPCR）检测方法。
通过生物信息学分析，我们选择了管圆线虫基因组ITS-2区域的一段序列，以确保引物和探针的特异性。我们从成虫寄生虫中提取DNA作为阳性对照，并使用DNeasy Blood&Tissue®试剂盒从幼虫中提取DNA。使用QuantiTect®试剂盒预混液在Smartcycler Cepheid®热循环仪上进行定量PCR反应。将来自人类、其他寄生虫和非洲蜗牛的DNA用作阴性对照。
阳性DNA对照的熔解曲线峰值分别为：广州管圆线虫21、哥斯达黎加管圆线虫22、血管圆线虫31。在阴性对照中，熔解曲线峰值为零。qPCR显示扩增效率为2（100%）。
在实验室中对三种具有临床意义的管圆线虫建立了标准化的多重qPCR检测方法。
