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

Of the clinical samples, 22.6% (14 of 62) were determined to be CPV-2 positive by the PCR-LFIA. The cutoff value (the mean value plus 3× the standard deviation for negative samples) of LFIA is 146 (Fig. 6) . The further analysis demonstrated the PCR-LFIA had a diagnostic agreement of 100% with PCR-GE (Table 4 ). All positive results were further confirmed by DNA sequencing to make sure that these are true positive isolates and there are no false-positive results. Sequences were edited using the SeqMan program and further verified by BLAST alignment tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These sequences of CPV-2 isolated from other countries or regions such as USA, New Zealand, India, Singapore, Shanghai and Yangzhou (available through GenBank) were used for comparison with the fourteen CPV-2 isolates. Multiple-sequence alignments were constructed by Table 3 Fluorescence signal values as a function of the copies of standard DNA measured by Nanoeasy 1700 Copies 3 × 10 8 3 × 10 7 3 × 10 6 3 × 10 5 3 × 10 4 3 × 10 3 3 × 10 2 3 × 10 1 3 × 10 0 NC* T 1 23,256 21,439 17,285 12,643 6802 1878 1031 165 28 0 T 2 22,138 18,948 16,859 10,853 8701 1206 929 93 66 11 T 3 24,359 using ClustalW method with the Lasergene MegAlign (DNASTAR, Madison, USA) software program (Fig. 7) . All sequences of fourteen positive samples were submitted to National Center for Biotechnology Information databases (GenBank accession numbers: MH614361-MH614374).