Evaluation of Methods for Extracting Viral DNA from Formalin-fixed Paraffin Specimens

Abstract

The following four methods for extracting viral DNA as templates for polymerase chain reaction (PCR) from formalin-fixed paraffin specimens (FFPS) were evaluated: phenol extraction and three commercial kits (Sepa-Gene, the DNA isolator PS kit, and DEXPAT). Extraction efficacies of these methods in materials from chickens infected with the chicken anemia virus (CAV) were compared by means of 2-step PCR. At 8, 14, and 22 days after inoculation, all methods detected PCR products of 279 bp. At 31 days after inoculation, however, products were detectable only in phenol-extracted FFPS. In the first step of PCR, when 480 bp was taken as the amplification region, products were detectable only in phenol-extracted FFPS collected at 8, 14, and 22 days after inoculation. When 279 bp was taken as the target sequence in the first step of PCR on the other hands, all the methods investigated detected CAV DNA. These results show phenol extraction to be most effective in extracting viral DNA from FFPS. But, it is time-consuming and requires the use of toxic reagents. In the light of these shortcomings, commercial kits too are useful since they successfully detect viral DNA from FFPS when 2-step PCR with a short target sequence is employed.