Evaluating DNA extraction kits for PCR detection of Aleutian mink disease virus

Abstract

The success of viral DNA amplification by PCR depends on the amount and quality of DNA, and is influenced by the extraction method, particularly when viral copy number is low or samples contain inhibitory substances. We compared four commercial nucleic acid extraction kits, Dynabeads viral extraction kit (DB) from Invitrogen, QIAamp DNA mini kit (QI) from Qiagen, MiniPrep kit (AX) from Axygen and the viral extraction kit (ZR) from Zymo Research for quality and yield of Aleutian mink disease virus (AMDV) DNA. The quality of extracted DNA was assessed by the success of PCR amplification and yield was measured by quantitative real-time PCR (qPCR). DNA from spleen and fecal samples of infected mink was extracted in triplicate and 45 PCR reactions were performed for each kit. Compared with DB, DNA extracted by QI, AX and ZR was amplified by PCR in 95.0 %, 53.3 % and 62.2 % of reactions, respectively, suggesting that DB and QI were comparable and outperformed AX and ZR. DB and QI kits were further compared by extracting DNA from identical samples in three trials. In trial 1, infected spleen, fecal matters, saliva swabs and plasma were used and viral recovery was measured by qPCR (64 reactions). In trial 2, spleen homogenate containing 10.5 × 10⁴ AMDV copies per μL was diluted 14 times in the range of 1 × 10° to 1 × 10^-4 and tested by PCR (435 reactions). In trial 3, DNA extracted from nine of the above dilution series was tested by qPCR (146 reactions). The mean viral copy number ranged between 31.2 and 10.5 × 10⁴ per μl of sample. DB and QI kits performed equally for viral recovery in all trials. The findings indicated that the extraction method influenced PCR amplification success, and DB and QI were comparable when input and output volumes were similar.