Detection of Ophiocordyceps sinensis and Its Common Adulterates Using Species-Specific Primers.
Detection of Ophiocordyceps sinensis and Its Common Adulterates Using Species-Specific Primers.
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使用物种特异性引物检测冬虫夏草及其常见掺杂物
DOI：
10.3389/fmicb.2017.01179
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发表时间：
2017
影响因子：
5.2
通讯作者：
Xiang L
中科院分区：
文献类型：
--
作者：
Liu Y;Wang XY;Gao ZT;Han JP;Xiang L
Ophiocorphinensis是一种感染蝠蛾科毛虫的真菌，使幼虫木乃伊化并产生特征子实体（基质），这些子实体被加工成最有价值的传统中药之一（TCM）。由于需求量很大，但供应量非常有限，该产品的价格非常高。与其他真菌掺假是一个常见的问题，需要测试制剂中是否存在正确的真菌。在本研究中，一种基于PCR的方法来鉴定O。基于一段内转录间隔区（ITS）序列，开发了一种新的中华绒螯蟹（sinensis）基因。片段大小为146 bp，即使在加工导致DNA片段化的材料中也可能被扩增。引物开发基于从总共89个O.冬虫夏草和潜在的混淆品以及GenBank中41种蛇果虫草和26种冬虫夏草的序列。用引物对DCF 4/DCR 4进行的检测表明，从O.中华子座，但不是来自来自掺假物的提取物。同时，还开发了用于检测古尼虫草、蝉虫草、蛹虫草、凉山虫草和垂穗蛇果虫草等常见伪品的特异性引物。本研究中开发的引物集合将有助于对声称仅含O的制剂进行鉴定。用于检测这些制剂中用作掺假物的真菌。
Ophiocordyceps sinensis is a fungus that infects Hepialidae caterpillars, mummifying the larvae and producing characteristic fruiting bodies (stromata) that are processed into one of the most valued traditional Chinese medicines (TCM). The product commands a very high price due to a high demand but a very limited supply. Adulteration with other fungi is a common problem and there is a need to test preparation for the presence of the correct fungus. In the current study, a PCR-based approach for the identification of O. sinensis based on a segment of the internal transcribed spacer (ITS) region was developed. The segments is 146-bp in size and is likely to be amplified even in materials where processing led to DNA fragmentation. Primer development was based on the alignment of sequence data generated from a total of 89 samples of O. sinensis and potential adulterants as well as sequences date from 41 Ophiocordyceps species and 26 Cordyceps species available in GenBank. Tests with primer pair, DCF4/DCR4, demonstrated generation of an amplicon from DNA extracted from O. sinensis stromata, but not from extracts derived from adulterants. Species-specific primer pairs were also developed and tested for detection of the common adulterants, Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, Cordyceps liangshanensis and Ophiocordyceps nutans. The collection of primers developed in the present study will be useful for the authentication of preparation claiming to only contain O. sinensis and for the detection of fungi used as adulterants in these preparations.