A novel non-sequencing approach for rapid authentication of Testudinis Carapax et Plastrum and Trionycis Carapax by species-specific primers

A novel non-sequencing approach was developed to detect short DNA fragments (ca 100 bp) for rapid authentication of two natural products, namely Testudinis Carapax et Plastrum and Trionycis Carapax, based on the difference in mitochondrial genome. Five specifically designed primer reactions were established to target species for reliable identification of their commercial products. They were confirmed to have a high level of inter-species-specificity and good intra-species stability. The limit of detection was estimated to be 1 ng of genomes for all of five assays. Also, the validation results demonstrated that the raw materials and processed products in addition to some of the highly processed products can be conveniently authenticated with good sensitivity and precision by this newly proposed approach. Especially, when reference sample mixtures were assayed, these primer sets have still performed well but not the prevailing COI barcoding technology. These could assist in the discrimination and identification of other animal-derived medicines for their form of raw material, the pulverized and the complex.


Introduction
Testudinis Carapax et Plastrum (TCP) and Trionycis Carapax (TC) are derived from the shell of Chinemys reevesii and Pelodiscus sinensis. They are not only important components in Chinese patent medicines (CPMs), but also are used for health supplements or functional foods. CPMs composed of TCP have a well-recognized curative effect in the treatment of osteoporosis, diabetic nephropathy, hypo-immunity, ageing, insomnia with sweating at night, etc. [1][2][3]. In addition, TC is an integral part of CPMs that are beneficial to cure cirrhosis, hepatitis, tuberculosis, Yin deficiency, as well as to inhibit tumour growth [4][5][6].
However, there are also numerous reports of fake or adulterant Chinese medicine, which has exposed consumers to a major public health risk and caused a non-negligible drug market disorder. Adulteration through the addition or substitution of similar substances in traditional Chinese medicine (TCM) is a type of medicine fraud defined as the intentional fraudulent modification of medicines to obtain a financial advantage. The fact that these two reptiles can be used for medicinal purpose only after their ages are above 3 years has caused an increasing scarcity of resources and economically motivated adulteration. On the other hand, similar morphological characteristics among species of close phylogenetic relationship and lack of professional experience can frequently lead to unintentional use of fake products. Moreover, both TCP and TC are processed in a manner where cutting, heating and sometimes even addition of vinegar are involved.
Accurate authentication of Chinese medicines is a strict legal requirement in many countries throughout America, Europe and Asia, and is a prerequisite for delivering a quality product that meets consumer expectations. Many efforts have been made to identify the origin of animal species in medicines and foods. Fourier transform infrared (FTIR), high-performance liquid chromatography (HPLC) and mass spectrometry (MS) have been employed to differentiate among species by spectral intensity and chromatographic behaviour, but similar chemical properties always make accurate identification of a mixture difficult [7][8][9][10][11]. Enzyme-linked immunosorbent assay (ELISA) is well recognized as a sensitive and robust technique for detecting low levels of original material from a species based on antibody and antigen reactions; however, species-specificity can be significantly compromised by high homology of protein sequences among animals and a high concentration of salt [12,13]. In recent decades, polymerase chain reaction (PCR)-based methods to verify the origin of a species have been considered the most preferred technology owing to the favourable specificity and stability of DNA fragments. For instance, the genome of ancient Egyptian mummies was used to research ancient human history and offered the perspective of deciphering Egypt's past [14]. Furthermore, DNA is present in most biological tissues and can be readily extracted from even a very small amount of test sample. Therefore, PCR-based methods are an ideal and powerful tool for identification of original material existing in final products [15,16].
DNA barcoding, particularly cytochrome c oxidase subunit 1 gene (COI) barcoding, has for a long time been most often used as an important means to identify ingredients and to detect spurious species, such as Cervi Cornu Pantotrichum (deer horn) and Serpentis Periostracum (snake slough) [17][18][19]. However, this method requires high purity of a DNA sample and cannot be applied to a mixture, such as adulterated products, or even medicinal materials slightly contaminated by other species. Moreover, COI universal DNA primers amplify a 710-bp fragment of the mitochondria, but the DNA from processed products is often severely degraded into very short fragments. So, for the analysis of these samples, the latter is a much more preferred target than the former. Hellebrand et al. [20] studied the influence of amplification length on test results, and it was found that the amplification of short fragments is more successful than that of longer fragments. On the other hand, intraspecific variation threshold is ambiguous owing to many factors including the interference of fluorescent dyes, base mismatching, evolutionary rate and so on. The divergence in a few cnidarians was far less than that typical for other animal phyla [21]. However, new primers designed to bind to highly conserved gene regions upstream of COI will aid the amplification of this gene region in species where standard primers fail, and will provide valuable information [13]. It is suggested that the threshold for intraspecific variation determined may be anything but convincing owing to various evolutionary rates and loci. Moreover, COI barcoding technology is of low capability in disturbance rejection, and it always undergoes a sequencing procedure of amplicon after PCR amplification. Accordingly, it was not commonly recommended that universal primers are used in DNA barcoding of processed material of Chinese medicine for species identification.
Mitochondrial DNA is applied for species identification because there are multiple copies per cell. The mitochondrial genome (mitogenome) in vertebrates consists of a circular DNA sequence of approximately 16-18 kb containing one control region, 22 tRNA sequences, two rRNA sequences and 13 peptide coding genes [22]. But the conservative areas of the species sometimes are not necessarily conserved in different individuals of the same species, and this has been leading to the failure of PCR amplification in some applications. Therefore, in this study, species-specific primers are particularly designed according to both intraspecific homology and interspecific variation in the mitochondrial complete genome of TCP, TC and their similar species. The aim was to establish a novel non-sequencing and reliable PCR-based approach that can be used for specific and rapid authentication of TCP and TC ( figure 1). In addition, these could also assist in the discrimination and identification of adulterants of other animal-derived Chinese medicines for their form of raw medicinal material, the pulverized and even the complex.

Materials
Three Testudinidae species including Chinemys reevesii, Trachemys scripta and Mauremys sinensis, and two Trionychidae species including Pelodiscus sinensis and Apalone ferox, were used in this study. Collection locations of these animals are listed in table 1, and all of the original samples were verified by COI barcoding. The tortoises and turtles were then handled to prepare the raw materials and processed products of their carapaces according to the relevant protocols recorded in the prevailing China Pharmacopoeia (Ch.P., 2015 edition) [23], as illustrated in figure 2. A total number of 64 commercial samples including raw materials, processed products and highly processed products were collected for species identification using the newly proposed approach. Three forms of traded commodities were collected from the TCM wholesale market, manufacturers and TCM hospitals as detailed in table 2.

DNA extraction
All solid samples were ground into their fine powder, and then subject to genomic DNA extraction by SDS-based protocols. In detail, 50 mg of the homogenized sample was mixed with 995 µl of extraction buffer (100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA, 0.5% (w/v) SDS) and 5 µl proteinase K (20 mg ml −1 ), and the mixture was incubated at 56°C for 6 h. After centrifugation at 12 000 r.p.m. for 15  Nucleic acid and protein spectrophotometry (BioSpec-mini, Shimadzu) were used to quantify the purity and concentration of the extracted DNA. These DNA samples extracted from raw materials or processed products were diluted to 50 ng µl −1 , and those from highly processed products were used directly as template in further PCR assays.   (Shanghai) Co., Ltd. Species-specific primer sets and their characteristics are summarized in table 3. Specificity testing with each primer set in the PCR assays was performed against five selected samples (table 1).  19.875 µl ultrapure water and 1 µl DNA template. The PCR cycler conditions used were an initial denaturation at 95°C for 3 min, followed by approximately 30-35 cycles of 95°C for 30 s, approximately 60-68°C for 30 s and 72°C for 1 min with a final extension at 72°C for 7 min. After resolution by 3% agarose gel electrophoresis and staining in ethidium bromide, the resulting amplicons were visualized under UV light. In order to verify the sequences of short-length fragments produced by the species-specific primers, PCR products were subjected to sequencing in both directions by Sangon Biotech (Shanghai) Co., Ltd.

Specificity and sensitivity
The verification of specificity was carried out under the optimum conditions for different batches of five Chelonia species. Sensitivity of the selected species-specific primers was determined in a concurrent PCR run with DNA template of a series of concentrations (10, 1, 0.1 and 0.01 ng µl −1 ) while the primer remained unchanged.        of one species mixed with the second species, with a total weight of 50 mg per sample. Individual samples were homogenized with 1 ml of SDS extraction buffer for DNA extraction by SDS-based protocols as aforementioned. Then, the selected primers and the optimized PCR conditions were applied to these reference samples.

Analysis of self-made samples and certified reference material
To investigate the scope of application of the newly proposed approach, 26 raw materials, 26 processed products, 26 highly processed products and a certified reference material of TCP (code: P; B/N: 121494-201604, National Institutes for Food and Drug Control, PRC) were analysed. The PCR assay was performed under optimized conditions using the species-specific primer sets.

Application of PCR assay to commercial products
As a next step, the developed method was used to assess the authenticity of 64 commercially available products, including both TCP and TC varieties, for the identification of animal origins and the verification of labelling compliance. Fifty milligrams of these samples were individually subjected to DNA extraction, and the optimized PCR conditions for each species were then applied to the sample extracts.

Screening of primer sets
Mitochondrial complete gene sequences from five species of Chelonia were incorporated to develop an accurate and rapid method for their identification. The specificity tests of the designed primers were predicted by the PRIMER-BLAST tool (table 4) and performed by uniplex PCR assay ( figure 4). When the primers were used for PCR amplification of genomic DNA extracted from five Chelonia species, PAF-1 does not amplify the target gene, and the designed primer sets except for PTS-1 and PTS-2 showed faint false-positive amplification at the corresponding location for the individual species. This false-positive amplification might be caused by improper PCR conditions. Consequently, PCR-2, PTS-2, PMS-1, PPS-2 and PAF-2, which resulted in stronger intensity bands without visible false-positive amplification, were selected for subsequent optimization of PCR conditions.

Optimized PCR conditions
Five primer sets were selected for the reliable identification of five Chelonia species and are listed in      primer, the type and amount of polymerase, annealing temperature and time, cycle times, temperature control method (two or three step) and the performance of equipment (data not shown). PCR conditions were optimized for the five species analysed (figure 5). The PCR cycler conditions used are summarized in table 5.

Specificity and sensitivity
The verification results of different batches of these five Chelonia species clearly demonstrated that each primer set produced a species-specific band without any visible non-specific bands ( figure 6). The amplicons were sequenced and edited to verify by BLASTn searches against the GenBank database ( figure 7). These primer sets were designed to identify different Chelonia species regardless of life stage. Sensitivity of a selected specific primer set for each of the five species was determined using one sample from each species. In all of the species, DNA concentrations of 10 and 1 ng µl −1 resulted in strong intensity bands ( figure 8).

Analysis of reference sample mixtures
To investigate whether the selected primers were applicable for adulterated products, 35 reference samples with known ingredient compositions were tested. As shown in figure 9, when these samples were analysed via PCR using selected specific primers, the corresponding species were successfully detected.

Analysis of self-made samples and certified reference material
The selected primers were applied to determine their scope in self-made samples and a certified reference material (TCP 121494-201403). As shown in figure 10, all of the raw materials and processed products were successfully analysed using the novel non-sequencing approach. Some of the highly processed products were not detected. This was likely due to the extensive processing that these products undergo. Species detection in the highly processed products may have been limited due to the processing as well as the presence of inhibitory ingredients present in these samples.

Application of PCR assay to commercial products
The ability of a novel non-sequencing approach to detect TCP and TC in commercial samples was tested with a variety of products, including raw materials, processed products and highly processed products