Abstract: To enable a rapid and sensitive determination of being virus-free for a Pseudostellaria heterophylla seedling, a nested RT-PCR method was developed. Detection of the presence of either Broad bean wilt virus(BBWV2) or Turnip mosaic virus(TuMV) in the sample was a proof of the existence of the diseases. Two pairs of degenerate primers were designed respectively according to the conserved sequence regions of the two coat protein (cp) genes of the viruses from GenBank. The nested RT-PCR detection protocols were optimized. Subsequently, detecting sensitivities of the conventional and the nested RT-PCRs were compared. Meanwhile, 7 samples of cultivated P. heterophylla along with 4 of virus-free test-tube plantlets were used in a challenge test on the nested RT-PCR methodology. The results showed that, by using the annealing temperature of 60℃ for BBWV2 and 62℃ for TuMV on the nested primers, specific bands could be detected on a 10⁵x dilution of the first strand cDNA for both viruses. That suggested at least 10-fold higher a sensitivity was achieved by the nested than the conventional RT-PCR. It was concluded that the newly established assay method was rapid, stable, sensitive and accurate for detecting BBWV2 or TuMV in P. heterophylla, and therefore, could be adequately applied to certify a virus-free status or not on a P. heterophylla sample.