Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

• Published in: Journal of Veterinary Medical Science Vol. 76; no. 4; pp. 509 - 516
• Main Authors: SUN, Yu-Ling, YEN, Chon-Ho, TU, Ching-Fu
• Format: Journal Article
• Language: English
• Published: Japan JAPANESE SOCIETY OF VETERINARY SCIENCE 01.04.2014; Japan Science and Technology Agency; The Japanese Society of Veterinary Science
• Subjects: Animals; Base Sequence; canine; Capsid Proteins - genetics; DNA Primers - genetics; Dog Diseases - diagnosis; Dog Diseases - virology; Dogs; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Enzyme-Linked Immunosorbent Assay - veterinary; lateral flow dipstick; loop-mediated amplification; Molecular Sequence Data; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - veterinary; Oligonucleotide Probes; Parvoviridae Infections - diagnosis; Parvoviridae Infections - veterinary; parvovirus; Parvovirus, Canine - isolation & purification; Sensitivity and Specificity; Sequence Alignment; Virology
• ISSN: 0916-7250; 1347-7439
• DOI: 10.1292/jvms.13-0448

Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection.