Authenticity Identification of the Traditional Chinese Herb Glycyrrhiza uralensis by Real-Time PCR and Loop-Mediated Isothermal Amplification

Background: Glycyrrhiza uralensis is a traditional Chinese herb, and sales volume of this herb is large. However, adulterant herbal materials threaten trade and consumer safety. MethodsA rapid detection system for the identification of G. uralensis was established using loop-mediated isothermal amplification (LAMP) and real-time fluorescence quantitative PCR (Real-Time PCR). The DNA was extracted and the G. uralensis primers were designed. LAMP and Real-Time PCR were performed to assessment the specificity of primers in species. The sensitivity of LAMP and Real-Time PCR was contrast by diluting DNA concentration gradient from 101 ng/μL to 10-5 ng/μL. Results: tThe results showed that LAMP and Real-Time PCR were specificity because G. uralensis were positive while similar species were not. The sensitivity of LAMP was similar to Real-Time PCR at DNA concentration of 10-4ng in 60 min. The results indicated that LAMP and Real-Time PCR are accurately, specificity and sensitivity in the Authenticity Identification of G. uralensis. Conclusions: LAMP is less time-consuming, convenient, and does not require expensive equipment. Thus, these findings suggested that a method system and standard of traditional Chinese herb market supervision should be established based on a DNA barcode sequence and LAMP for authenticity identification. for method. The present study reported for the first time the use of LAMP for authenticity identification of the traditional Chinese herb, G. uralensis . The present findings indicated that a method system and standard of TCH market supervision should be established based on DNA barcode sequence and LAMP for authenticity identification


Background
Traditional Chinese herbs (TCHs) play an important role in disease treatment and studies as indicated the clinical efficacy of TCHs against certain diseases [ 1 ]. G. uralensis is a well-known and well-regarded traditional Chinese medicine. G. uralensis is often used as a cough expectorant that effectively relieves sore throats due to its anti-inflammatory and anti-allergy effects.
The quality of TCHs is important for the effect and safety. Some defective goods and falsified herbs are entering the TCHs market due to resource depletion and profit-driven influences, which poses a serious risk to public health. However, it has been recently reported the adulterant drugs cause life-threatening poisonings [2][3][4][5]. Therefore, the problem of authenticity identification of Chinese medicinal materials has become an important issue in improving the quality of TCHs and guaranteeing import, export, and human health [ 6 ].
The traditional identification and shape classification method do not meet the needs for identification of TCHs, especially for broken up into small pieces after complicated processing, which have disadvantages, including insufficient effects and accuracy [ 7 ]. In addition, none of these methods can be used to identify the closely related species with obviously similar chemical and morphological characteristics [ 8 ].
LAMPand Real-Time PCR are rarely used in the identification of TCH because they are often use for the identification of microorganism [9][10][11][ [12][13][14][15]. In the present study, a real-time LAMP protocol was developed, and the reliability and sensitivity of this protocol was investigated for authenticity identification of traditional Chinese herbs using the ITS2 gene as the DNA barcoding gene.

Traditional Chinese herbs
Four Chinese herbs with similarity to G. uralensis were used as negative samples to test for specificity (Table 2), considering the following characteristics. First, the difficulty in distinguishing them from G. uralensis based on shape, smell and colour when TCHs are processed into small pieces or powder due to these herbs were similar to G. uralensis. Second, these herbs had similar therapeutic effects on disease. However, these herbs were less expensive than G. uralensis but also less effective. Lastly, detection of G.
uralensis by the Entry-Exit Inspection and Quarantine Bureau has generally found these herbs adulterated.
No specific permits were required for these activities because the G. uralensis and negative samples used in this study were obtained from an orchard, which is a those from relevant species has tested. And such a test be used to better address the specificity. LAMP Primers were designed using PrimerExplorer software [ 8 ]. LAMP was performed using a Loopamp kit with Fluorescent detection reagent (Eiken Chemical Co. Ltd., Japan), incubated at 65°C for 90 minutes and then at 80°C for 2 min in a Loopamp real-time turbidimeter (LA-500, Teramecs Co. Ltd., Japan).

Real-Time PCR Specificity analysis
The PCR efficiency of ITS2 and psbA-trnH sequences has compared in 400 TCHs samples, and it showed that the success rate of ITS2 and psbA-trnH sequences is 92.8% [ 8 ]. Thus, ITS2 was selected after filtering the barcoding sequences (Table 1)

Specificity of LAMP
Three primers groups of LAMP were designed and preliminarily selected, and the best group was selected according to the initial turbidity time. Four primers (B3, F3, BIP and FIP) required by LAMP and six different distinct sequences of target DNA identified were shown in figure 2 (Fig. 2). For primer group 2, turbidity occurred after 20 minutes, and the curve decreased after 50 minutes. LAMP pyrophosphate detection by white turbidity after the precipitation peak caused a reduction in the precipitation amount, which decreased the detection value. For primer group 1, turbidity occurred after 60 minutes, while a detection line did not show up for the other group until the end (Fig. 3). Thus, primer group 2 was selected as the optimal group for LAMP, and loop primers where then designed according to the reaction time. The LAMP was considered positive when lines appeared after 20 minutes for G. uralensis without lines appearing for the other similar species (Fig. 4). Therefore, these findings indicated that the LAMP is specific to G.
uralensis. The early appearance in turbidity with the addition of loop primers indicated that the addition of loop primers accelerates the LAMP reaction. The turbidity appearance early with add loop primers indicated the loop primers accelerates the LAMP.
We perform the LAMP Specificity test of the mixing the DNA from G. uralensis with those from relevant species. And such a test to better address the specificity for authenticity identification of G. uralensis (Fig. 5).

Sensitivity between Real-Time PCR and the LAMP
The DNA template was diluted in concentration gradient of 10 1 -10 -5 ng/µL. The detection concentration limit in the Real-Time PCR for herbal DNA was 10 -4 ng/μL (Fig. 6). The Ct value was greater than 30 when the DNA concentration was 10 -5 ng/μL and The Ct value increased with dilution of DNA. Because sensitivity of Real-Time PCR was higher which can detect a lower concentration of DNA than conventional PCR.
The sensitivity of Real-Time PCR was compared to that of LAMP using the same DNA template. Turbidity was lower with the reduction of DNA concentration. However, no turbidity was detected when DNA concentration was 10 -5 ng/μL (Fig. 7a), which was no change when the amplification time varied 20~ 35 minutes. Moreover, no amplification was observed when the DNA concentration was lower than 10 -4 ng/μL (Fig. 7b), which validated sensitivity of LAMP at 10 -4 ng/μl. Ethics approval and consent to participate Not applicable. This article does not contain any studies with human participants or animals performed by any of the authors. No specific permits were required for these activities because the G. uralensis used in this study were obtained from an orchard, which is a demonstration base of Minzu University of China.

Consent for publication
The authors agree for publication.

Competing interests
No conflict of interest exits in the submission of this manuscript, and no any competing financial and/or non-financial interests in relation to the work described. At the same time, manuscript is approved by all authors for publication.  Screening the best group of LAMP primers. Screening for the best group of LAMP primers for G. uralensis by real-time turbidity.