An Improved Polymerase Cross-Linking Spiral Reaction Assay for Rapid Diagnostic of Canine Parvovirus 2 Infection

Xin Xu; Xueyu Wang; Wen Hu; Qianqian Wu; Lunguang Yao; Yunchao Kan; Jun Ji; Yingzuo Bi

doi:10.3389/fvets.2020.571629

An Improved Polymerase Cross-Linking Spiral Reaction Assay for Rapid Diagnostic of Canine Parvovirus 2 Infection

Front Vet Sci. 2020 Oct 30:7:571629. doi: 10.3389/fvets.2020.571629. eCollection 2020.

Authors

Xin Xu^{1

2}, Xueyu Wang¹, Wen Hu¹, Qianqian Wu¹, Lunguang Yao^{1

2}, Yunchao Kan¹, Jun Ji¹, Yingzuo Bi³

Affiliations

¹ Henan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Nanyang Normal University, Nanyang, China.
² Collaborative Innovation Center of Water Security for Water Source Region of Mid-line of South-to-North Diversion Project of Henan Province, School of Agricultural Engineering, Nanyang Normal University, Nanyang, China.
³ College of Animal Science, South China Agricultural University, Guangzhou, China.

Abstract

With increasing complications of canine parvovirus infection cases, disease diagnosis and treatment have become more difficult. In this study, specificity primers for the conserved region of the VP2 gene of canine parvovirus 2 (CPV-2) were synthesized and evaluated. An improved polymerase cross-linking spiral reaction (PCLSR) method for early and rapid diagnosis of CPV-2 was established. The results showed that the amplification reaction was optimal when run at 62°C for 50 min and could be used to detect CPV-2 without any cross-reactions with other pathogens of canine infectious diseases. Reaction results were directly judged by the naked eyes, with the positive amplification tube shown as luminous yellow and the negative tube as bright purple. Compared with the previously reported polymerase spiral reaction (PSR) method for CPV-2 detection, this reaction was performed using improved primer pairs and a better dye identification method (using an indicator comprising phenol red and cresol red). The detection limit of PCLSR was 3.9 × 10¹ copies using gel electrophoresis or a visible dye. The positive rate of 132 clinical samples was 42.42%, which was identically the same as that of the PSR method and slightly higher than that of the colloidal gold strip method (39.39%). The newly developed CPV-PCLSR assay shows the advantage of rapid visualization of results and offers a convenient and rapid method for early CPV-2 diagnosis with higher sensitivity and specificity than the established methods.

Keywords: canine parvovirus; detection method; polymerase cross-linked spiral reaction; simplicity; visible detection.