Introduction

Canine parvovirus (CPV) is an epidemic enteric pathogen of dog worldwide (1). Owing to highly contagious nature of infection, a rapid and early diagnosis is essential (2). In this study, we designed a highly specific and sensitive diagnostic system for CPV using the Loop-mediated isothermal amplification (LAMP) method (3), compared to a PCR and validated its merits.

Materials and methods

DNA was extracted from CPV strain (ATCC VR953) and 50 canine diarrheic samples suspected of suffering. CPV infection by the DNAzol (MRC, INC). For LAMP, a set of 4 primers, 2 outer and 2 inner primers, were designed from CPV genomic DNA. Reaction time and temperatures were optimized for 60 min at 65 °C, respectively. PCR was performed to identify 249 bp fragments situated on the VP2 gene. The sensitivity and specificity were compared for these 2 methods.

Results

A large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture of LAMP. The presence or absence of this white precipitate allows easy detection of the amplification of CPV genomic DNA without gel electrophoresis. The detection limit using the LAMP method was up to 1 fg of DNA, when compared to 10 fg of DNA by PCR The sensitivity of LAMP in comparison with that of PCR was 92% and the specificity was 100% (k=0.76).

Conclusions

The advantage of the LAMP method is speed; only 2 hours were necessary for diagnosis of CPV infection. Therefore, this assay should be a useful aid to diagnosis of CPV infection in dogs, suggesting that this method may achieve clinical application.

References

1. Carmichael et al., 1981, Adv Vet Sci Comp Med, 25:1-37.

2. Pereira et al., 2000, Vet Microbiol , 75:127-133.

3. Notomi et al., 2000, Nucleic Acids Res 20:1691-1696.