Subscribe to RSS
DOI: 10.1055/s-0043-113448
DNA Barcoding for Industrial Quality Assurance
Publication History
received 10 March 2017
revised 16 May 2017
accepted 27 May 2017
Publication Date:
29 June 2017 (online)
Abstract
DNA barcoding methods originally developed for the identification of plant specimens have been applied to the authentication of herbal drug materials for industrial quality assurance. These methods are intended to be complementary to current morphological and chemical methods of identification. The adoption of these methods by industry will be accelerated by the introduction of DNA-based identification techniques into regulatory standards and monographs. The introduction of DNA methods into the British Pharmacopoeia is described, along with a reference standard for use as a positive control for DNA extraction and polymerase chain reaction (PCR). A general troubleshooting chart is provided to guide the user through the problems that may be encountered during this process. Nevertheless, the nature of the plant materials and the demands of industrial quality control procedures mean that conventional DNA barcoding is not the method of choice for industrial quality control. The design of DNA barcode-targeted quantitative PCR and high resolution melt curve tests is one strategy for developing rapid, robust, and reliable protocols for high-throughput screening of raw materials. The development of authentication tests for wild-harvested Rhodiola rosea L. is used as a case study to exemplify these relatively simple tests. By way of contrast, the application of next-generation sequencing to create a complete profile of all the biological entities in a mixed herbal drug is described and its potential for industrial quality assurance discussed.
Key words
authentication - Rhodiola rosea - Phyllanthus amarus - Ocimum tenuiflorum - next-generation sequencing - metabarcoding - Crassulaceae - LamiaceaeSupporting Information
- Supporting Information
Five examples of the authorsʼ unpublished works have been used to illustrate this review. These are as follows.
Example 1: The BP DNA-based identification method for O. tenuiflorum; Example 2: The BPNARM; Example 3: A specific qPCR assay for R. rosea; Example 4: A HRM curve assay for Rhodiola species; Example 5: NGS assay of Phyllanthus samples. The experimental details (Materials and Methods, Results) for each example are included as Supporting Information.
There are two supporting tables.
Table 1S: Commercial “holy basil” samples used for development of the BP DNA-based identification method for O. tenuiflorum; Table 2S: Rhodiola ITS sequences used in this study.
There are ten supporting figures.
Fig. 1S: Multiple alignment of trnH-psbA region sequences from holy basil commercial samples; Fig. 2S: Bases 190 – 260 of a multiple alignment of trnH-psbA region sequences from holy basil commercial samples; Fig. 3S: Bases 300 – 370 of a multiple alignment of trnH-psbA region sequences from holy basil commercial samples; Fig. 4S: HPTLC of Rhodiola samples and reference compounds; Fig. 5S: HPLC trace of an extract from R. rosea, showing the presence of the rosavine marker compounds rosarin, rosavin, and rosin; Fig. 6S: Two Rhodiola ITS regions selected for HRM curve analysis primers design; Fig. 7S: HRM curve analysis results using the HRM1, HRM2, and HRM3 primer pairs with R. rosea and R. crenulata templates; Fig. 8S: HRM curve analysis results using the HRM1, HRM2, and HRM3 primer pairs with 11 different Rhodiola templates; Fig. 9S: Clarity dPCR results with species-specific primers; Fig. 10S: Representation of the constructs produced for NGS barcoding.