Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2

Zhuang, Linlin; Ji, Yongxin; Tian, Peilong; Wang, Kaixuan; Kou, Chengkun; Gu, Ning; Zhang, Yu

To establish the optimal conditions for PCR-LFIA, positive control (CPV-2-N1 strain) and negative control (PBS) were used for determining both amplified products and primer dimers and demonstrating the effect of PCR products purification ( Fig. 3 ; Table 1 ). As shown in Fig. 3 , the PCR-GE method confirmed a correct amplification target of 253-bp fragment. Only some primer dimers were observed in the negative control. In addition, the effect of PCR products purification was simultaneously verified by 1.5% agarose gel electrophoresis. The bands of positive control remained bright but the primer-dimer bands disappeared in negative control (Fig. 3a) . Furthermore, the results of PCR-LFIA were then confirmed by Nanoeasy 1700. Correspondingly, the positive control displayed a specific characteristic peak of test line and showed a slight decrease with purification. However, the negative control showed no specific characteristic peak and its fluorescence value was significantly reduced with purification (below the test strip's cutoff value of 62) (Fig. 3b) . The results verified the PCR method and the effect of purifying PCR products based on magnetic beads, which would effectively overcome the obstacle of false positive results.