Development and Application of TaqMan-MGB-Probe-Based Quantitative Real-Time Polymerase Chain Reaction for Rapid Detection of Dictyocaulus filaria

Zhengqin Gao^*Jin Xing

• National Institutes for Food and Drug Control (China), Beijing, Beijing Municipality, China

Lungworm disease caused by Dictyocaulus filaria is an infectious disease of sheep and goats all over the world in recent years, which causes huge economic losses and are considered as potential public health threats. To date, there have been few studies on Dictyocaulus filaria, its pathogenic mechanism is still unclear, and there are no effective vaccines or drugs to prevent and control the disease. Therefore, it is necessary to develop a rapid and reliable molecular diagnostic method to aid in the study of this novel parasite. Here, we developed a TaqMan-MGB-probe-based quantitative real-time polymerase chain reaction (qPCR) for rapid detection of Dictyocaulus filaria for the first time. Specific primers and TaqMan-MGB-probe were designed targeting the ITS2 region of Dictyocaulus filaria. The assay showed a strong specificity for detecting Dictyocaulus filaria, and had no cross-reactivity with other control pathogenic parasites. The assay had high sensitivity with the lower limit of quantification (LLOQ) and the limit of detection (LOD) are both 1.5 copies per reaction. The assay exhibited excellent repeatability and reproducibility of the intra-assay coefficient of variation (CV) of 0.11–0.58% and the inter-assay CV of 0.33–2.19%. Finally, the developed TaqMan-MGB-probe-based qPCR was used to detect 200 clinical samples. The results indicated that the positive detection rate of Dictyocaulus filaria was 3.5% (7/200), and the result showed good consistency with conventional PCR. The developed TaqMan-MGB-probe-based qPCR assay has the advantages of high sensitivity and strong specificity, high throughput, convenient and fast, and cost-effectiveness. It is of great significance to safeguard animal quality and human health.