For PCR-positive controls, recombinant plasmids containing APM DNA (kindly provided by Didier Raoult, Unite des Rickettsies, Universite de la Mediterranee, Marseille, France) were constructed. Primer pairs bracketing the L396 and R596 genes were used to amplify 1,560bp and 879-bp full gene regions, respectively, using 300 nmol/L of forward primers 396 F (5′-TTA ATC ATC TTC CAA AAA ATT TAA TTC-3′) and 596 F (5′-ATG TCG TTA TCA AAA CAA GTA GTT CC-3′), and 300 nmol/L of reverse primers 396 R (5′-ATG GCG AAC AAT ATT AAA ACT AAA A-3′) and 596 R (5′-CTA ATT TTC AAT ATA GTG CGT AGA TTC TA-3′). These PCR products were purifi ed by using the QIAquick Gel Extraction Kit (QIAGEN) and then cloned into a pCR-II TOPO vector by using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA). Recombinant plasmids were then isolated by using the QIAprep Spin Miniprep Kit (QIAGEN) and quantifi ed by UV spectroscopy. Standard curves were prepared from serial 10-fold dilutions of the quantifi ed plasmid in nuclease-free water containing 100 μg/mL of herring sperm DNA (Promega, Madison, WI, USA).