Identification of Lonicera japonica by PCR-RFLP and allele-specific diagnostic PCR based on sequences of internal transcribed spacer regions.

The flower buds of Lonicera japonica Thunb. are widely used in Chinese medicine for their anti-inflammatory prosperities. Some closely related species of L. japonica which have similar morphology but weaker biological activity are also used medicinally. The discrimination of L. japonica from its adulterants is currently limited to methods of morphology and chemical fingerprinting. The analysis of sequence variation in the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) has recently become an effective method for identification of medicinal herbs. In the present study, we found that nrDNA ITS sequences differ significantly between L. japonica and the 4 tested adulterant species. We identified a distinctive site which can be digested by restriction endonuclease ECO52 I and designed a pair of allele-specific primers for diagnostic PCR on the nrDNA ITS2 region. These two techniques provide accurate, effective, and rapid approaches to distinguish L. japonica from closely related species.