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.