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

The genomic DNA of CPV-2-N1 strain was extracted for PCR amplification. PCR products were purified by Magbeads PCR Purification Kit (Nanoeast, Nanjing, China). The PCR amplification products were ligated into the pUC57 vector by using Hieff Clone™ Zero TOPO-TA Cloning Kit (Yeasen, Shanghai, China) after purified and cloned in Escherichia coli. Positive clones were identified by sequencing (GenScript, Nanjing, China). The positive clones were cultured and the plasmids were extracted using Magnetic Plasmid Extraction Kit (Nanoeast, Nanjing, China), and the content of the plasmids was determined by Nano-300 Micro Spectrophotometer, then the plasmids were stored at − 20°C. The plasmid copy number was calculated following the formula [34] : copies/ μL = (6.02 × 10 23 ) × (ng/μL × 10 − 9 ) / (DNA length × 660) by using the plasmid pUC57 containing the desired fragment as a standard. Standard DNA is diluted from 3 × 10 8 to 3 × 10 0 copies/μL for PCR amplification to evaluate the detection limit of PCR-LFIA.