Rapid identification of cervus antlers by species-specific PCR assay

Abstract A rapid PCR technology was developed to differentiate Cervus antlers species and adulteration based on the difference in mitochondrial genome. Three specifically designed primer sets were confirmed to have high inter-species specificity and good intra-species stability. Limits of detection were estimated to be 1 ng of genomes for reindeer and 10 ng for the other species. Especially, when the mixture of Cervus antlers and reindeer or sambar was assayed, these primer sets still exhibited strong capability of differentiation but not the conventional COI barcoding. By using the newly developed approach, five batches out of fourteen commercial Cervus antler products were identified to be fake products made from reindeer antlers. It has shown its good potential to be extensively applied in the identification of counterfeits or adulterates of Cornu Chinese medicines for their pulverized and processed form, and even the traditional Chinese patent medicines composed of these species. Graphical Abstract


Introduction
Cervus antler is a famous and popular tonic derived from the ossified antlers or exfoliated antler base of Cervus elaphus Linnaeus (CEL) or Cervus nippon Temminck (CNT) (Chinese Pharmacopoeia Committee 2015). This medicine has been widely used for debilitated persons as it has significant effects to warm and tonify kidney, as well as to vitalize and improve health in clinical practices (Zha et al. 2011). In addition, it can activate blood circulation, disperse blood stasis and reduce swelling, which is quite beneficial for patients after surgery and gynecology treatment.
As Cervus antler is very limited in resources, fake products or adulterations of Cervus family in commercial Cervus antler products were prevalent, which have been mentioned in previous studies (Kim et al. 2012;Wang et al. 2009;Zha et al. 2011). For example, antlers of other Cervidae animals, such as reindeer (Rangifer tarandus Linnaeus, RTL) and sambar (Rusa unicolor Kerr, RUK), were used to obtain illegal economic profits. On the other hand, similar morphological characteristics and lacking professional experience make it difficult to distinguish the species of closer phylogentic relationship.
Many methods including HPLC fingerprint, electrophoresis-based and biological MSbased technologies have been developed for the analysis of deer products (Gao et al. 2010;Guo et al. 2016;Huang et al. 2017;Yang et al. 2015Yang et al. , 2017. Complicated procedures and expensive equipments are usually needed, and accurate identification of a mixture can't be readily achieved due to similar chemical properties. Recently, DNA barcoding has made a great contribution to the molecular identification of traditional Chinese medicine (Chen et al. 2013;Ludt et al. 2004). For instance, cytochrome c oxidase subunit 1 (COI) barcoding was commonly used in the authentication of animalderived Chinese medicine, such as raw Bungarus parvusi and velvet antler, even processed deer products Liu et al. 2014;Zhang et al. 2011). However, this method is not suitable for DNA samples of lower purity or the mixtures of various animal origins, such as adulterated products or medicinal materials slightly contaminated by other species. Meanwhile, COI barcoding is also not efficient in disturbance rejection, and complicated procedures for DNA sequencing of amplification products are indispensable. What's more, the original DNA is often degraded into very short fragments after the medicines were processed. Previous studies have shown that the short fragments are more favorable for amplification (Hellebrand et al. 1998). Accordingly, the species-specific PCR with high specificity is an alternative choice, which has been successfully used in authentication of Testudinis Carapax et Plastrum and Trionycis Carapax, and Bombyx batryticatus, etc (Tang et al. 2007;Yang, et al. 2018aYang, et al. , 2018b.
In this study, species-specific primers were designed according to both intraspecific homology and interspecific variation in mitochondrial complete genome of CEL, CNT and their similar species. The aim was to establish a novel non-sequencing and reliable PCR-based approach that can be utilized for specific and rapid authentication of Cervus antlers products.

Primer design and screening
The specificity assays of the designed primers predicted by Primer-BLAST tool were performed by uniplex PCR. The results showed that three primer sets coded as A, C and E (Table S3) presented stronger intensity bands at 187 bp, 200 bp and 101 bp individually under pre-set amplification conditions of low annealing temperature and more cycle numbers ( Figure S2). Subsequently, these three primer sets were re-coded as PC, PRT and PRU respectively for amplification of Cervus antlers, reindeer and sambar (Table S4), and they were applied in further optimization of PCR conditions.

Optimized specific PCR conditions
The specific PCR conditions were then optimized. Effects of the cycle number, primers concentration and annealing temperature were studied ( Figure S3). As a result, all amplification products from three pairs of primer (PC, PRT and PRU) were found after the DNA templates of CEL, CNT, RTL and RUK have been amplified for 33 reaction cycles respectively. The optimum concentration of PC was 0.4 mM, while the PRT and PRU were both 0.2 mM. The best annealing temperature of PC, PRT, and PRU were 64 C, 63 C, and 61 C respectively. Other conditions, such as concentration of MgCl 2 , amount of dNTP and DNA template, and the unit of Taq polymerase were kept consistent with 1.4 PCR Amplification under Experimental session of Supplementary Material. The relevant information of optimized PCR conditions was summarized in Table S5.

Specificity and sensitivity
The verification results of different batches of these antler species adequately demonstrated that each primer set produced its own species-specific band, in other words, three primer sets PC, PRT and PRU amplified corresponding species respectively without any other visible non-specific bands under optimal PCR conditions ( Figure S4). Meanwhile, the amplicons were sequenced and verified by BLASTn searches through the GenBank database. As presented in Figure S5, the amplicon sequences obtained by bidirectional sequencing can be aligned with the corresponding species. In addition, the amplified regions and the number of bases were in accord with the theoretical parameters, which suggested that the specific primers used in this study were reliable and feasible. The sensitivity analysis was then carried out, clear bands have been achieved after the PCR amplification against CEL, CNT and RUK while the DNA concentration of these species was 10 ngÁmL À1 , however the band intensity observed after the amplification by PRU was much weaker than PC. Meanwhile, PRT was even able to generate a bright band against 1 ngÁmL À1 of RTL DNA template ( Figure S6).

Analysis of reference sample mixtures
As shown in Figure S7, twenty reference sample mixtures of different proportions were successfully detected by corresponding specific primers via PCR. The results showed that the band intensity presented an increasing or decreasing trend with component ratio varied, and the obtained bands after amplification by PC and PRT were of similar intensity. In general, the amplified bands were relatively clear, which has also indicated that three primer sets could be applicable for the identification of adulterated products.

Application of PCR assay to commercial products
The results of non-sequencing species-specific PCR performed on commercial products (Table S2) could be seen in Figure S8 and were summarized in Table S6. According to the results, all DNA extracted from 14 batches of antler products were amplified by selected primer sets. Obviously, 9 out of 14 batches (6 $ 14) of commercial products were successfully amplified by primer set of CNT and CEL, while the other five batches (1 $ 5) of these products were amplified solely by PRT, however, none of 14 batches has been amplified by PRU. The results above indicated that all of the fake commercial products were made of RTL antler but not RUK according to the results of PCR assays.

Conclusions
In our study, the results demonstrated that species-specific primers can rapidly and accurately identify the antler species, and compared to the DNA barcoding technology, it is much more time saving and low cost. It was also found that the antler of reindeer has been used to make fake Cervus antler more often than sambar. In conclusion, the novel non-sequencing approach described in this study, proved to be simple, time-saving, low-cost, accurate, reliable and sensitive, although a validation step for amplicon sequencing may be needed to ensure accuracy in practice. Our technique could be important from an economic point of view in terms of fair trade and consumer rights and could be considered as a further improvement of traditional method-based assay for the identification of deer antler products.

Disclosure statement
The authors declared no conflict of interest.

Funding
This study was supported by National Natural Science Foundation of China (81773855), Six Talent Peaks (YY-010) and Qinglan Project (2016).