Identification of Lonicera japonica by PCR-RFLP and Allele-Specific Diagnostic PCR Based on Sequences of Internal Transcribed Spacer Regions

Abstract

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.

Key words

Lonicera japonica - Caprifoliaceae - nrDNA ITS region - PCR‐RFLP - allele‐specific - diagnostic PCR

References

• 1 The State Pharmacopoeia Commission of the PRC .Pharmacopoeia of China, Part 1. Beijing; Chemical Industry Press 2005: 152-153
• 2 Wang C Z, Li P, Ding J Y, Fishbein A, Yuan C S. Discrimination of Lonicera japonica THUNB. from different geographical origins using restriction fragment length polymorphism analysis.  Biol Pharm Bull. 2007;  30 779-782
• 3 Xie Y Q. The influence of the modification of “Pharmacopoeia of The People's Republic of China” on Lonicera japonica planting.  Da Zhong Ke Ji. 2007;  8 146
• 4 Liu X F, Li L, Leng C H. Comparison of the content of chlorogenic acid and galuteolin among different origins of Honeysuckle and Shan Yin Hua.  Subtropical Crop Communications of Zhejiang. 2007;  29 20-21
• 5 Shaw P C, Wang J, But P P H. Authentication of Chinese medicinal materials by DNA technology, 1st edition. Hackensack; World Scientific Publishing 2002: 229-231
• 6 Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue.  Phytochem Bull. 1987;  19 11-15
• 7 White T J, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Innis M, Gelfand D, Sninsky J, White T PCR protocols: a guide to methods and application. San Diego; Academic Press 1990: 315-322
• 8 Don R H, Cox R T, Wainwright B J, Baker K, Mattick J S. “Touchdown” PCR to circumvent spurious priming during gene amplification.  Nucleic Acids Res. 1991;  19 4008