[Identification of Cordyceps cicadae and Tolypocladium dujiaolongae based on ITS sequences and chemical pattern recognition method].

ABSTRACT

Based on ITS sequences, the molecular identification of Cordyceps cicadae and Tolypocladium dujiaolongae was carried out, and high-performance liquid chromatography(HPLC) fingerprint combined with chemical pattern recognition method was established to differentiate C. cicadae from its adulterant T. dujiaolongae. The genomic DNA from 10 batches of C. cicadae and five batches of T. dujiaolongae was extracted, and ITS sequences were amplified by PCR and sequenced. The stable differential sites of these two species were compared and the phylogenetic tree was constructed via MEGA 7.0. HPLC was used to establish the fingerprints of C. cicadae and T. dujiaolongae, and similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminant analysis(PLS-DA) were applied to investigate the chemical pattern recognition. The result showed that the sources of these two species were different, and there were 115 stable differential sites in ITS sequences of C. cicadae and T. dujiao-longae. The phylogenetic tree could distinguish them effectively. HPLC fingerprints of 18 batches of C. cicadae and 5 batches of T. dujiaolongae were established. The results of CA, PCA, and PLS-DA were consistent, which could distinguish them well, indicating that there were great differences in chemical components between C. cicadae and T. dujiaolongae. The results of PLS-DA showed that six components such as uridine, guanosine, adenosine, and N~6-(2-hydroxyethyl) adenosine were the main differential markers of the two species. ITS sequences and HPLC fingerprint combined with the chemical pattern recognition method can serve as the identification and differentiation methods for C. cicadae and T. dujiaolongae.