Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction

Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of the putative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplification of CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by using a 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNA sequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by a guanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involving phenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany, Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of the PCR-amplified DNAs with the enzymes HaeIII, HinfI, and HpaII indicated that the 14 CAV isolates can be assigned to seven groups, with isolates from different countries usually exhibiting the greatest number of restriction site differences.