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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 63  |  Page : 410-415  

Rapid identification of medicinal leech by species-specific polymerase chain reaction technology


Department of Chinese Medicine and Pharmacy, School of Pharmacy, Jiangsu University, Zhenjiang 212013, China

Date of Submission27-Nov-2018
Date of Decision14-Jan-2019
Date of Web Publication16-May-2019

Correspondence Address:
Huan Yang
School of Pharmacy, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, Jiangsu Province
China
Yuping Shen
School of Pharmacy, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, Jiangsu Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_596_18

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   Abstract 


Background: Leech is a well-known animal-derived Chinese medicine; intentionally or unintentionally, interfusing fake products have exposed patients to high risk. Objective: In this study, we would like to find a rapid and accurate technology to identify medicinal leech. Materials and Methods: Three sets of species-specific primer were designed, and polymerase chain reaction (PCR) conditions were optimized for the four species analyzed. Specificity and sensitivity test were carried out, and the reference sample mixtures were analyzed. Finally, the developed method was used to assess the authenticity of commercially available products. Results: Three sets of species-specific primer were confirmed to have high interspecies specificity and good intraspecies stability. The limit of detection was estimated to be 1 ng for all of four assayed species. Subsequent validation demonstrated that the raw material, processed, and even highly processed products of leech can be conveniently authenticated with good sensitivity and precision by this newly established PCR-based method. Conclusion: These designed novel primer sets have shown distinct performance on the identification of adulterate products. The new technology needs no procedure for sequencing and has been successfully employed for the reliable authentication of raw material of leech and even their processed and highly processed products.

Keywords: Authentication, deoxyribonucleic acid barcoding, leech, polymerase chain reaction, species-specific primer


How to cite this article:
Zheng Y, Lu B, Yang Y, Jiao Z, Chen L, Yu P, Shen Y, Yang H. Rapid identification of medicinal leech by species-specific polymerase chain reaction technology. Phcog Mag 2019;15:410-5

How to cite this URL:
Zheng Y, Lu B, Yang Y, Jiao Z, Chen L, Yu P, Shen Y, Yang H. Rapid identification of medicinal leech by species-specific polymerase chain reaction technology. Phcog Mag [serial online] 2019 [cited 2019 Dec 15];15:410-5. Available from: http://www.phcog.com/text.asp?2019/15/63/410/258397



Summary

  • A novel polymerase chain reaction technology was developed to determine leech species based on the difference in mitochondrial genome. Three species-specific primers were designed and confirmed to have a high level of interspecies specificity and good intraspecies stability. The limit of detection was estimated to be 1 ng of genomes for all of four assays. The raw material and processed products can be conveniently authenticated and with both good sensitivity and precision by this newly proposed approach. Especially, these primer sets have still performed well when the samples are adulterated.




Abbreviations used: DNA: Deoxyribonucleic acid; COI: Mitochondrial cytochrome c oxidase subunit 1; PCR: Polymerase chain reaction; bp: Base pair.


   Introduction Top


Leech, a well-known animal-derived Chinese medicine, is the dry product of the whole body of Whitmania pigra Whitman, Hirudo nipponica Whitman, or Whitmania acranulata Whitman. Medically, it has been widely used as an anticoagulation and antithrombosis agent in clinics for a very long time.[1] In addition, it is also considered as promising drug targets for anticancer and anti-inflammation therapy.[2],[3] Medicinal leeches appear to provide methods of reducing blood coagulation and reliving venous pressure from pooling blood, especially after plastic and organ transplantation surgery.[4],[5] However, the abundance requirement and high price of leech in market have caused serious intentional fraudulent modification of this medicine, and the similar morphological characteristics in different species of closer phylogenetic relationship and lack of professional experience can usually lead to unintentionally interfusing fake products. For instance, Poecilobdella manillensis Lesson was misused as medicinal leech quite often, and study had shown that it has different effects on thrombin and coagulation pathways with medicinal leech.[6] Owing to accurate authentication of herbal medicines which is strictly a legal requirement worldwide and a prerequisite to deliver quality products that meet consumers' expectations on their efficacy,[7] highly selected and sensitive methods for leech accurate identification are vital.

Conventional methods for leech identification are based on the morphological characteristics of the intact adult leech which is not applicable for the larval and incomplete individuals yet. Several current analysis methods are mostly relying on chromatographic and mass spectrometer (MS)-based techniques which require high cost, complicated, and expensive instrument.[8],[9],[10] For instance, high-performance liquid chromatography fingerprint and gas chromatography (GC)/MS were popularly employed for detection of characteristic small molecule, and electrophoresis-based or biological MS-based technologies were often used to monitor signature proteins or specific peptide fragments.[11],[12],[13],[14],[15] However, similar chemical properties always make accurate identification of a mixture difficult. In addition, complicated procedures have to be performed while using these technologies and expensive equipment is usually needed. At present, deoxyribonucleic acid (DNA) molecular identification that can differentiate species correctly in close phylogenetic relationship groups for herbal medicines has been well recognized and become more and more popular.[16],[17],[18],[19],[20],[21] It always undergoes a sequencing procedure of amplicon after polymerase chain reaction (PCR) amplification using universal primers, and the obtained sequence is compared with that in database to identify species. For instance, the DNA barcodes system was established for the reliable authentication of Chinese medicinal plants.[22] However, this method requires high purity of DNA sample and not suitable for adulterants. Furthermore, the broad applications of DNA barcoding are limited as the original DNA is probably degraded into very short fragments after Chinese medicines have been highly processed. Alternatively, species-specific primers have been designed to bind to highly conserved gene regions to aid the amplification of this gene region in species where universal primers fail.[23] Up to date, they have been successfully used to identify Agkistrodon acutus, Zaocys dhumnades, and so on to demonstrate their good prospects for the identification of Chinese animal medicines.[24],[25],[26],[27],[28],[29],[30] However, there are currently no correlative studies on the molecular identification of leech species using species-specific PCR.

In this study, a reliable PCR-based approach was established for the identification of leech species for the first time. Species-specific primers were designed and evaluated by DNAMAN, Oligo and online NCBI Primer-BLAST, followed by optimization of amplification conditions and validation for specificity and sensitivity [Figure 1]. The newly developed method can be used for specific and rapid authentication of leech products for their form of raw material, the processed, and even the highly processed.
Figure 1: Flow chart for the establishment of the novel strategy

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   Materials and Methods Top


Samples

Three leech species including W. pigra, H. nipponica, or W. acranulata and a fake leech species P. manillensis were used in this study. Collection locations of these animal origins were listed in [Table 1], and all of them used cytochrome c oxidase subunit I barcoding for verification and authenticated by Professor Jun Chen (Pharmacognosy Research Facility, School of Pharmacy, Jiangsu University). The samples were then handled to prepare the raw materials and processed products of leech according to the relevant protocols recorded in the prevailing China Pharmacopoeia (Ch. P., 2015 Ed, Vol. 1) as illustrated in [Figure 2]. In addition, a certified reference substance (raw material of W. pigra; B/N: 121061-201305) was purchased from National Institutes for Food and Drug Control, PRC.
Table 1: Sources of Leech species used in the study

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Figure 2: Procedures for assessing raw materials, processed, and highly processed products of leech

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Primers' design

Mitochondrial complete gene sequences from four species of leech were incorporated to develop an accurate and convenient method for their identifications. All the primers were designed in accordance with the principles including length between 18 base pair (bp) and 25 bp, GC% >40%, and no hairpin structure and dimers. Species-specific primer sets were designed according to both intraspecific homology and interspecific variation in mitochondrial genome of leeches and their similar species using Oligo software (Version 7.60). All of them were evaluated by Oligo and online NCBI Primer-BLAST [Figure 3] and synthesized by Sangon Biotech (Shanghai) Co., Ltd. As two species, namely W. pigra and W. acranulata have a similarity of 95% in genome, no specific primers can be employed to distinguish them. Eventually, they shared one primer set in this study, and the length of its target product is 63 bp. In addition, two pairs of primers were selected for H. nipponica and P. manillensis, respectively, and the expected amplicons for each species were 102 bp and 75 bp. The three species-specific primer sets were listed in [Table 2]. Specificity testing for each primer set in the PCR assays was performed on the four selected species.
Figure 3: Flow chart for the design of species-specific primer sets

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Table 2: Primer sets used for polymerase chain reaction assay in this study

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Deoxyribonucleic acid extraction and polymerase chain reaction amplification

All solid samples after complete lyophilization were grounded into their fine powder and then subjected to genomic DNA extraction by SDS-based protocols according to the procedures provided by Yang et al.[7] Nucleic acid and protein spectrophotometer (Bio Spec-mini, Shimadzu) were used to quantify the purity and concentration of the extracted DNA. These DNA samples extracted from raw materials, processed, and highly processed products were diluted to 100 ng/μL as template in further PCR assays. PCR amplification was performed in a final reaction volume of 25 μL composed of buffer, Mg 2+, dNTP, primers, Taq polymerase, DNA template, and H2O. During the amplification, several factors including the concentration of template and primer, the type and amount of polymerase, annealing temperature, and time and cycle times may affect the output. Based on the result of preliminary experiments, the annealing temperature of the primers was optimized. The PCR cycler conditions used were an initial denaturation at 95°C for 3 min, followed by 30–35 cycles of 95°C for 30 s, 60°C~64°C for 30 s, and 72°C for 1 min with a final extension at 72°C for 5 min. After resolution of the resulting amplicons by electrophoresis on 2%~3% agarose gel and staining in ethidium bromide, the gel was put under ultraviolet light for visualization of bands.

Specificity and sensitivity

The specificity test was carried out under the optimum conditions for different batches of leech. Sensitivity of the selected species-specific primers was determined in a concurrent PCR run with DNA template of a series of concentrations (0.001 ng/μL, 0.01 ng/μL, 0.1 ng/μL, 1 ng/μL, 10 ng/μL, and 100 ng/μL) while the concentration of the primer remained unchanged.

Analysis of reference sample mixtures

Before making various reference mixtures, sample of different leech species was collected to undergo DNA extraction and serve as a positive control. Three reference mixtures (WP:P, WA:P, and HN:P) were prepared in five proportions (9:1, 7:3, 1:1, 3:7, and 1:9) of one species mixed with the second species, with a total weight of 20 mg per sample. Individual sample was homogenized with 1 mL of the extraction buffer for DNA extraction by SDS-based protocols as aforementioned. The selected primer sets and the optimized PCR conditions were then applied to these reference sample mixtures.

Application of polymerase chain reaction assay to commercial leech products

Finally, the developed method was used to assess the authenticity of commercial products listed in [Table 3], for the identification of animal origins and the verification of labeling compliance. Ten milligram of each of these samples was individually subjected to DNA extraction. The optimized PCR conditions for each species were then applied to the DNA samples.
Table 3: Traded leech products from different sources tested in the study

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   Results Top


Optimized polymerase chain reaction conditions

As shown in [Figure 4], under the conditions where the cycle number was 35, species-specific primers could not only amplify the corresponding species but also other species when the annealing temperature is low. Primers achieved a good specificity when the temperature was raised to the appropriate level, and the optimal annealing temperature of the PW, PHN, and PP was 62°C, 60°C, and 61°C, respectively. The PCR conditions employed were summarized in [Table 4].
Figure 4: Optimized polymerase chain reaction conditions for four species (M: Low ladder, SN127)

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Table 4: Optimized polymerase chain reaction conditions for three primers analyzed in this study

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Specificity and sensitivity

Many samples were used to verify the specificity of these primers. As shown in [Figure 5], the extracted DNA from W. pigra and W. acranulata has been amplified against the same primer (PW) with the product at 63 bp while the amplification of the DNA from H. nipponica and P. manillensis produced two bands at 102 bp and 75 bp. The verification results of different batches of these four-leech species adequately demonstrated that each primer set can produce a species-specific band without any other bands for all of the negative controls. These primer sets were successfully used to specifically identify different leech species regardless of life stage or morphology. Sensitivity of a selected specific primer set for each of the four species was determined on one batch of the collected sample of each species, and DNAs within the range from 1 pg to 100 ng were subjected to the PCR amplification. The resulting bands after amplification of DNAs from these leech species are shown in [Figure 6]. The brightness of the strip gradually decreased with the decline of DNA concentration. Finally, it was found that the detection limits were 10 ng for W. acranulata and 1 ng for W. pigra, H. nipponica, and P. manillensis, indicating that the detection of four leeches by these primers has a fairly high sensitivity.
Figure 5: Specificity test for three specific primer sets

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Figure 6: Sensitivity test for three specific primer sets. The concentration of template deoxyribonucleic acid from lane A to lane F was 100, 10, 1, 0.1, 0.01, and 0.001 ng/μL

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Analysis of reference sample mixtures

To investigate whether the three primers were applicable for adulterated products, 15 reference samples with known ingredient compositions were assayed. As shown in [Figure 7], the DNA mixtures have been amplified, and each species in all of the 15 mixed samples was accurately detected without interference. The specificity of the primers was not affected while being applied to the identification of the adulterated leeches, and the specific primers only amplified the corresponding species in the mixed DNAs. The brightness of the strip was also obvious even though the adulteration ratio was 10%, and it is consistent with the results of previous sensitivity test. These results indicated the good feasibility and suitability of the primers for the detection of both leech species and adulterated leech products by the developed technology.
Figure 7: Analysis of reference sample mixtures by polymerase chain reaction

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Application of polymerase chain reaction assay to commercial products

The selected primers were applied to commercial products including raw processed and highly processed leech. As shown in [Figure 8], all of the raw and processed leeches were successfully identified using the novel nonsequencing approach. Twelve batches of commercial samples were authenticated as true leech products made from W. pigra or W. acranulata, while another three batches were counterfeited by P. manillensis. None of H. nipponica was found in commercial leech products. This could be due to the lack of natural resources and higher price. In addition, three batches (L12, L13, and L14) of these products did not show positive reaction against any of three primer sets (PW, PHN, and PP), indicating that they were fake products made from other animal origins rather than P. manillensis. Meanwhile, one batch (L15) was amplified by both PW and PP, and it has been considered as an adulterant product. It can be seen that the situation of counterfeiting or misusing is very serious as the percentage of fake or adulterate products detected was higher than one-third, and it warned the public to be vigilant while they consume leech products.
Figure 8: Analysis of commercial product of leech by polymerase chain reaction

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   Discussions Top


In this study, three specific primers were designed for rapid identification of four leech species. In the optimized PCR conditions, the result of amplification of each species is consistent with the target DNA fragment. Moreover, all DNA samples at the concentration of 10 ng/μL have resulted in single band of strong intensity and demonstrated that the primers have good specificity and high sensitivity. In addition, it can accurately identify not only individual species but also adulterated leeches, which was verified while being applied to commercial products. The fake or adulterate leech products were more than one-third of all tested samples, indicating the importance of accurate authentication and market regulation of commercial leech products. In our study, the result showed that species-specific primers can usefully, rapidly, and accurately identify the leech species. Compared with the DNA barcoding technology, it saves a lot of time and cost; with an unknown species DNA, we can confirm it within 3 h. Moreover, conservative areas of the species sometimes are not necessarily conserved in different individuals of the same species and that has been leading to the failure of PCR amplification in some applications, it can be avoided by using species-specific primer. However, in this study, W. pigra Whitman and W. acranulata Whitman were not differentiated; however, it does not negatively affect the authentication as both of them are genuine. In the future study, we would focus on looking for specie-specific primers based on other DNA barcode such as the internally transcribed spacer or other biological technologies such as proteomics.


   Conclusion Top


This established novel approach is convenient, low cost, accurate, 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 will be very useful for the inspection of edibility and medicinal value of leech.

Acknowledgements

We would like to thank Dr. Mingqi Wang, School of Pharmacy, Jiangsu University, for his valuable advice on manuscript preparation.

Financial support and sponsorship

This study was financially supported by the National Natural Science Foundation of China (81773855), the China Postdoctoral Science Foundation (2017T100338), Six Talent Peaks Project in Jiangsu (YY-010), and Qing Lan Project (2016).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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