Loop-mediated isothermal amplification for the detection of goose circovirus.
BACKGROUND
Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection's clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection.


RESULTS
The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRed(TM) solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods.


CONCLUSIONS
The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.
Department of Poultry Viral Diseases, National Veterinary Research Institute, Partyzantów 57 Avenue, 24-100, Puławy, Poland. grzegorz.wozniakowski@piwet.pulawy.pl
Grzegorz
G
Woźniakowski
Wojciech
W
Kozdruń
Elżbieta
E
Samorek-Salamonowicz
D000818:Animals; D052160:Benzothiazoles; D018173:Circoviridae Infections / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D005777:Geese; D025202:Molecular Diagnostic Techniques / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009930:Organic Chemicals / Q000378:metabolism; D011044:Poland; D011201:Poultry Diseases / Q000175:diagnosis* / Q000821:virology; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013696:Temperature; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I