Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

Mingming Zhao,Lan Wu,Licheng Guo,Wei Liu,Wei Sun,Song Yan,Yuhua Shi,Chao Xiong,Shilin Chen

doi:10.1038/srep25370

Abstract

Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided.

Highlights

IntroductionPrevious methods used to authenticate saffron have involved physical, chemical, and molecular fields, such as 1H NMR metabolite fingerprinting[7], two-dimensional gas chromatography[8], SCAR markers[9,10,11], and melting curve analysis[6]
Daucus carota[6], Zea mays[6], and Nelumbo nucifera[6]
Our results showed that no variation at the site of the internal transcribed spacer 2 (ITS2) sequence between the original plant C. sativus and the dry herb saffron

Summary

Introduction

Previous methods used to authenticate saffron have involved physical, chemical, and molecular fields, such as 1H NMR metabolite fingerprinting[7], two-dimensional gas chromatography[8], SCAR markers[9,10,11], and melting curve analysis[6]. These methods can identify saffron and its adulterants more quickly and accurately than traditional morphological detection. The ITS2 region has proven to be a core DNA barcode for authentication of a broader range of medicinal plant taxa with high variability and species-discrimination[26] These characteristics make ITS2 a good bar-code gene choice for LAMP-specific primer design[27]. This study sought to establish a simple, rapid, and sensitive method to distinguish saffron from its adulterants by using LAMP based on the ITS2 sequence and to provide a practical SOP model for herbal medicine detection

Methods

Results

Conclusion