Primer concentrations and annealing temperature are alsoimportant for achieving high specificity and efficiency of the RT-PCRamplification (Ma and Michailides, 2007; Wei et al., 2009). Reduc-ing primer concentrations from 0.2 M to 0.1–0.14 M excludedthe non-specific amplification in the multiplex RT-PCR in this study.The optimized multiplex RT-PCR was specific and sensitive fordetecting different targets of TBTD in a single reaction.