目的 建立小儿肺咳颗粒中鳖甲DNA分子鉴定方法.方法 根据鳖和佛罗里达鳖线粒体全基因序列的差异设计特异性引物PP2和PA3,采用CTAB法、酚仿抽提法提取、纯化DNA,并进行特异性引物扩增和方法学考察,产物以2％琼脂糖凝胶电泳检测.结果 鳖及佛罗里达鳖的DNA经扩增后分别在183、95 bp处产生条带,且未观察到非特异性扩增条带,引物PP2及PA3对两者DNA的检测限分别为0.10、0.010 ng/μL.有4批小儿肺咳颗粒中检出鳖DNA,未检出佛罗里达鳖DNA;6批产品两者DNA均未能检出.结论 该方法方便、 可靠地鉴定小儿肺咳颗粒中鳖甲,有助于控制含鳖甲成方制剂的质量.

Background: Testudinis Carapax et Plastrum (TCP, Guijia) and Trionycis Carapax (TC, Biejia), which made from the shell of Chinemys reevesii (CR) and Pelodiscus sinensis (PS), were two kinds of widely used animal-derived Traditional Chinese medicine (TCM). Some cheap substitutes such as the carapace and plastron of Trachemys scripta (TS), Mauremys sinensis (MS), or Apalone ferox (AF) shells obtained from restaurants are sometimes used, which expose the public health to a high risk and cause unfair competitions in the market. Objective: The objective of the study was to develop a multiplex polymerase chain reaction (PCR) approach to simultaneously differentiate five Chelonia species and identify adulteration of two natural products. Materials and Methods: Five novel species-specific primers were designed for CR, TS, MS, PS, and AF, followed by optimization of PCR conditions and validation for specificity and sensitivity. Then, a deliberate mixture was analyzed to verify the capability of adulteration detection. Finally, it was used to examine commercial products. Results: The developed method proved to be highly specific and detection limit was 1 ng for all the species tested. Particularly, it was still applicable and reliable when processed products or deliberate adulteration were examined. Two batches of commercial raw TCP products were identified to be counterfeited by TS using the newly proposed approach. Conclusion: The newly proposed multiplex PCR method showed sufficient merits to be readily employed as a regular means to authenticate edible and medicinal TCM products made of TCP and TC.

Background: Asini Corii Colla (ACC; donkey-hide glue) and Taurus Corii Colla (TCC; bovine-hide glue) are popular healthcare food supplements as well as well-recognized medicine in Traditional Chinese medicine clinics. Due to large demand of them and limited resources of their animal origins, the skin of other animals are often used to make their fake or adulterated products. Objective: In this study, we aimed to develop a species-specific polymerase chain reaction (PCR) approach to detect deoxyribonucleic acid (DNA) fragments of ACC and TCC for rapid authentication purpose. Materials and Methods: Four sets of novel species-specific primer were designed, and PCR conditions were optimized for the PCR assay, which was further validated for specificity and sensitivity. Then, deliberate mixture was analyzed to further verify the capability of adulteration detection by the developed PCR method. Finally, it was used to assess the authenticity of commercial products. Results: Four primers' sets were specific for amplification of extracted mitochondrial DNA of donkey, bovine, swine, and horse when the annealing temperature was 59 degrees C, 59 degrees C, 63 degrees C, and 65 degrees C, and 40 thermal cycles was performed. Under this optimized PCR conditions, 1 ng/mu L of horse DNA template has been detected in the sensitivity test, with 0.1 ng/mu L for donkey, bovine, and swine. The assay was capable of detecting spiked species at 10% level in premixed ACC or TCC samples. Conclusion: The newly developed PCR-based technology was specific and sensitive, and it was a convenient approach for the authentication of commercial ACC and TCC products.

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.

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.

A multiplex PCR assay was developed to simultaneously differentiate four antelope species and identify adulteration in Cornu Saigae Tataricae. Four novel primer sets were designed with high inter-species specificity and intra-species stability. Limit of detection was estimated to be 10 ng of genomes. When a mixture of antelope hornand fake species was assayed, it exhibited powerful differentiation capability. 5 out of 12 batches of commercialproducts were identified to be counterfeited or adulterated with Ovis aries Linnaeus and/or Capra hircus Linnaeus. It is readily applicable in routine analysis for identification of sham or adulterants of Cornu Saigae Tataricae.

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.

A novel PCR technology was developed to detect short DNA fragments using species-specific primers for rapid and non-sequencing authentication of Bombyx batryticatus based on differences in the mitochondrial genome. Three specifically designed primer reactions were established to target species for the reliable identification of their commercial products. They were confirmed to have a high inter-species specificity and intra-species stability. The limit of detection was estimated as 1 ng of genomes for Beauveria bassiana and 100 pg for Bombyx mori and Metarhizium anisopliae. Furthermore, validation results demonstrated that raw materials and their processed products can be conveniently authenticated with good sensitivity and precision using this newly proposed approach. In particular, when counterfeits were assayed, these primer sets performed well, whereas COI barcoding technology did not. These could also assist in the discrimination and identification of adulterates of other animal-derived medicines in their pulverized and processed forms and even in complexes.

目的 基于特异性扩增建立动物药龟甲与鳖甲的真伪鉴定技术.方法 根据龟甲与鳖甲及3种常见伪品的全基因组序列的差异设计了5对特异性引物(PCR、PTS、PMS、PPS、PAF),采用SDS法、酚仿抽提法从龟甲与鳖甲中提取与纯化DNA,并通过聚合酶链式反应(PCR)对目标片段进行扩增,产物以琼脂糖凝胶电泳分析及凝胶成像系统检测,并根据电泳条带的有无及分子量大小以鉴定真伪.结果 龟甲与鳖甲及其常见伪品巴西龟、花龟、佛罗里达鳖的DNA经扩增后分别在120、120、81、105、114 bp处产生条带,且引物间无交叉反应,体现出很强的专属性,5个物种DNA的检出限为1 ng.自制药材、饮片、掺伪品均扩增出与目的片段相一致的条带.在市售样品中,18批龟甲和20批鳖甲经鉴定为正品,2批龟甲药材经鉴定为来源于巴西龟的伪品.结论 本文所建立的技术可准确、快速地鉴定龟甲与鳖甲的真伪,有助于监控其产品质量.