{"title":"利用基于 PCR-CRISPR/Cas12a 的核酸检测技术鉴别 Fritillaria Bulbus 的物种","authors":"","doi":"10.1016/j.jarmap.2024.100589","DOIUrl":null,"url":null,"abstract":"<div><div><em>Fritillaria</em> Bulbus (FB) is a traditional Chinese herbal medicine known for its efficacy in relieving coughs and alleviating asthma. It is frequently used in conjunction with other <em>Fritillaria</em> species due to their highly similar morphological characteristics. Given their considerable medicinal and economic value, convenient and accurate methods for identifying these herbal medicines are essential. This study introduces a nucleic acid detection method that integrates PCR amplification of the target region of nuclear ribosomal DNA with CRISPR/Cas12a mediated trans-cleavage of a fluorescent reporter. This method distinguishes between the two most commercially valuable species of FB, specifically <em>Fritillaria Cirrhosa</em> Bulbus (FCB) and <em>Fritillaria Ussuriensis</em> Bulbus (FUB). A conserved fragment of nuclear ribosomal DNA was chosen as the target sequence for designing crRNAs specific to each species. Both crRNAs exhibit high sensitivity in detecting amplified genes, with a detection limit of 3.0 ng/μl. No cross-reactivity was detected with non-target species, indicating high specificity<em>.</em> The practicality of this method was validated through the analysis of standard medicinal materials and real-world samples. Compared to DNA barcoding, this method exhibited superior capability in detecting mixed samples, thereby establishing a benchmark for the application of CRISPR/Cas-based nucleic acid detection in verifying the authenticity of traditional Chinese medicinal materials.</div></div>","PeriodicalId":15136,"journal":{"name":"Journal of Applied Research on Medicinal and Aromatic Plants","volume":null,"pages":null},"PeriodicalIF":3.8000,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Species discrimination of Fritillaria Bulbus using PCR-CRISPR/Cas12a-based nucleic acid detection\",\"authors\":\"\",\"doi\":\"10.1016/j.jarmap.2024.100589\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div><em>Fritillaria</em> Bulbus (FB) is a traditional Chinese herbal medicine known for its efficacy in relieving coughs and alleviating asthma. It is frequently used in conjunction with other <em>Fritillaria</em> species due to their highly similar morphological characteristics. Given their considerable medicinal and economic value, convenient and accurate methods for identifying these herbal medicines are essential. This study introduces a nucleic acid detection method that integrates PCR amplification of the target region of nuclear ribosomal DNA with CRISPR/Cas12a mediated trans-cleavage of a fluorescent reporter. This method distinguishes between the two most commercially valuable species of FB, specifically <em>Fritillaria Cirrhosa</em> Bulbus (FCB) and <em>Fritillaria Ussuriensis</em> Bulbus (FUB). A conserved fragment of nuclear ribosomal DNA was chosen as the target sequence for designing crRNAs specific to each species. Both crRNAs exhibit high sensitivity in detecting amplified genes, with a detection limit of 3.0 ng/μl. No cross-reactivity was detected with non-target species, indicating high specificity<em>.</em> The practicality of this method was validated through the analysis of standard medicinal materials and real-world samples. Compared to DNA barcoding, this method exhibited superior capability in detecting mixed samples, thereby establishing a benchmark for the application of CRISPR/Cas-based nucleic acid detection in verifying the authenticity of traditional Chinese medicinal materials.</div></div>\",\"PeriodicalId\":15136,\"journal\":{\"name\":\"Journal of Applied Research on Medicinal and Aromatic Plants\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.8000,\"publicationDate\":\"2024-10-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Applied Research on Medicinal and Aromatic Plants\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2214786124000627\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Applied Research on Medicinal and Aromatic Plants","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214786124000627","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
Species discrimination of Fritillaria Bulbus using PCR-CRISPR/Cas12a-based nucleic acid detection
Fritillaria Bulbus (FB) is a traditional Chinese herbal medicine known for its efficacy in relieving coughs and alleviating asthma. It is frequently used in conjunction with other Fritillaria species due to their highly similar morphological characteristics. Given their considerable medicinal and economic value, convenient and accurate methods for identifying these herbal medicines are essential. This study introduces a nucleic acid detection method that integrates PCR amplification of the target region of nuclear ribosomal DNA with CRISPR/Cas12a mediated trans-cleavage of a fluorescent reporter. This method distinguishes between the two most commercially valuable species of FB, specifically Fritillaria Cirrhosa Bulbus (FCB) and Fritillaria Ussuriensis Bulbus (FUB). A conserved fragment of nuclear ribosomal DNA was chosen as the target sequence for designing crRNAs specific to each species. Both crRNAs exhibit high sensitivity in detecting amplified genes, with a detection limit of 3.0 ng/μl. No cross-reactivity was detected with non-target species, indicating high specificity. The practicality of this method was validated through the analysis of standard medicinal materials and real-world samples. Compared to DNA barcoding, this method exhibited superior capability in detecting mixed samples, thereby establishing a benchmark for the application of CRISPR/Cas-based nucleic acid detection in verifying the authenticity of traditional Chinese medicinal materials.
期刊介绍:
JARMAP is a peer reviewed and multidisciplinary communication platform, covering all aspects of the raw material supply chain of medicinal and aromatic plants. JARMAP aims to improve production of tailor made commodities by addressing the various requirements of manufacturers of herbal medicines, herbal teas, seasoning herbs, food and feed supplements and cosmetics. JARMAP covers research on genetic resources, breeding, wild-collection, domestication, propagation, cultivation, phytopathology and plant protection, mechanization, conservation, processing, quality assurance, analytics and economics. JARMAP publishes reviews, original research articles and short communications related to research.