Real-time PCR assays for APM were developed from multiple primers and probes designed for conserved re-gions of class I NCLDV genes L396 and R596, class III NCLDV gene L65, as well as the R656 gene, from the published APM genome sequence (GenBank accession no. NC_006450) by using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). All probes were labeled at the 5′ end with 6-carboxy-fl uorescein and quenched at the 3′ end with Black Hole Quencher-1 (Biosearch Technologies, Novato, CA, USA). Different primer and probe combinations were evaluated, and the 2 PCR assays that gave the best performance were selected for further studies (Table 1 ). Assays were performed by using the iQSupermix Kit (Bio-Rad, Hercules, CA, USA) in 25-μL reaction volumes. Amplifi cation was performed on an iCycler iQReal-Time Detection System (Bio-Rad) by using the following cycling conditions: 95ºC for 3 min for 1 cycle; 95ºC for 15 s and 55ºC for 1 min for 45 cycles each. Total nucleic acid was extracted from all specimens by using either the NucliSens Automated Extractor (bioMérieux, Boxtel, the Netherlands) or the automated BioRobot MDx (QIAGEN, Valencia, CA, USA) according to the manufacturers' instructions. Each clinical specimen was also tested for the human ribonuclease P gene to measure nucleic acid integrity as previously described (5) .