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

Abstract

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.

A multiplex TaqMan real-time PCR for detection and differentiation of four antigenic types of canine parvovirus in China

“…Several assays have been reported for the detection or quantitation of CPV DNA [12][13][14][15][16][17][18], including PCR, nested PCR, iiPCR, RPA, LAMP-ELISA, LAMP-LFD, LAMP, polymerase spiral reaction, and SYBR Green based real-time PCR, but none of these assays enable differentiation CPV antigenic types and CPV-like viruses (MEV and FPV). Several other assays have been reported for the detection and differentiation of type 2-based vaccines and field strains of CPV [19] or typing of three antigenic types of CPV [20] or CPV and MEV [21] assay and MGB probe real-time RT-PCR, but none of these assays enables simultaneous detection and differentiation of four antigenic types of CPV.…”

Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2

“…The CPV LFS RPA reaction tubes were held in a closed fist for 15 min, and the results were inspected directly with the naked eye within 5 min. As were most of the molecular methods for detection of CPV-2, such as PCR [15], nested PCR [13], real-time PCR [12], iiPCR [16], and LAMP [11,14], the assay was developed based on the VP2 gene. Although the point mutations in the VP2 protein have been associated with CPV types, the full consideration of the CPV variants were taken into account when designing the primers and probe, and the highly conserved region of the VP2 gene was set as the template.…”

“…A series of gene amplification-based assays have been developed for CPV-2, such as polymerase chain reaction (PCR), nested PCR, real-time PCR, loop-mediated isothermal amplification (LAMP) and insulated isothermal PCR (iiPCR), which have played an important role in the control of CPV-2 infection [11][12][13][14][15][16]. However, due to the requirements of an expensive thermocycler, a centralized laboratory facility and experienced technicians, implementation of the PCR assays is limited at the point-of-need (PON) diagnosis.…”

“…Compared to the PCR assays, the use of isothermal technologies reduces the need for high precision instrumentation and consistent electrical power. Although the previously developed LAMP assays do not require specialized equipment, they are difficult to design as 4-6 primers are required [11,14]. A report of the iiPCR method was described its sensitive detection of CPV-2, but the assay was dependent on a specialized instrument, POCKIT™ Nucleic Acid Analyzer [16].…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Lateral flow dipstick (LFD) was developed as well for simultaneous detection of pathogenic Leptospira spp. (Nurul Najian et al 2016), measles virus (Xu et al 2016), animal babesiosis, caused by Babesia bovis and Babesia bigemina , foot-and-mouth diseases (Waters et al 2014), Japanese encephalitis (Deng et al 2015), P. vivax, and P. falciparum (Yongkiettrakul et al 2014;Kongkasuriyachai et al 2017), Jembrana disease virus (Kusumawati et al 2015), canine parvovirus (Sun et al 2014), Vibrio alginolyticus (Plaon et al 2015), Macrobrachium rosenbergii nodavirus and shrimp yellow head virus (Khunthong et al 2013).…”