The melting temperatures (Tm) of each PCR product was determined by melting curve analysis using LightCycler 480 gene scanning software (version 1.5) (Roche Applied Science). The cycle number (Cn), representing the target sequence copy number, was taken to be the number of PCR cycles needed for the change in fluorescence signal of the amplicons to exceed the detection threshold value. The sensitivity and specificity values were calculated and expressed using the method described previously [25].