ABSTRACT
The molecular identification of Ophiocordyceps sinensis and its adulterants was carried out by real-time fluorescent PCR with TaqMan probe. Genomic DNA was extracted from 100 samples of Ophiocordyceps sinensis and its adulterants. MEGA 7.0 software was used for comparative analysis to define the variable sites between Ophiocordyceps sinensis and its adulterants according to the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). A set of specific primers and TaqMan probe were designed using Primer Premier 6.0 software, and sensitivity and specificity studies were performed on two different real-time fluorescent PCR systems (Genesig q16 and Bio-Rad CFX96). The sensitivity study showed that the detectable DNA template concentration of Ophiocordyceps sinensis for the real-time fluorescent PCR was 0.016 ng·μL-1 in the Bio-Rad CFX96 system and 15.527 ng·μL-1 in the Genesig q16 system, respectively. Meanwhile, this method had good specificity for Ophiocordyceps sinensis on Genesig q16 and Bio-Rad CFX96 systems, so Ophiocordyceps sinensis could be clearly distinguished from Ophiocordyceps nutans, Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae, Cordyceps liangshanensis, Cordyceps gracilis. Our results indicate that real-time fluorescent PCR with TaqMan probe can be used to accurately identify Ophiocordyceps sinensis from its adulterants. This provides a technical method that has wide applications for market management and quality control of Chinese materia medica.
ABSTRACT
To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.
Subject(s)
Breeding , DNA, Fungal , Genetics , DNA, Intergenic , Genetics , Genes, Mating Type, Fungal , Hypocreales , Chemistry , Classification , Genetics , PhylogenyABSTRACT
To accelerate the breeding process of cultivated Ophiocordyceps sinensis and increase its yield, it is important to identify molecular fingerprint of dominant O. sinensis. In the present study, we collected 3 batches of industrially cultivated O. sinensis product with higher yield than the others and compared their internal transcribed spacer (ITS) sequences with the wild and the reported. The ITS sequence was obtained by bidirectional sequencing and analyzed with molecular systematics as a DNA barcode for rapid and accurate identification of wild and cultivated O. sinensis collected. The ITS sequences of O. sinensis with detailed collection loci on NCBI were downloaded to construct a phylogenetic tree together with the sequences obtained from the present study by using neighbor-joining method based on their evolution relationship. The information on collection loci was analyzed with ArcGIS 10.2 to demonstrate the geographic distribution of these samples and thus to determine the origin of the dominant samples. The results showed that all wild and cultivated samples were identified as O. sinensis and all sequences were divided into seven phylogenetic groups in the tree. Those groups were precisely distributed on the map and the process of their system evolution was clearly presented. The three cultivated samples were clustered into two dominant groups, showing the correlation between the industrially cultivated samples and the dominant wild samples, which can provide references for its optimized breeding in the future.
Subject(s)
Breeding , DNA, Fungal , Genetics , DNA, Intergenic , Genetics , Genes, Mating Type, Fungal , Hypocreales , Chemistry , Classification , Genetics , PhylogenyABSTRACT
Objective To clone the full-length of cDNA protein marker cyanase (IP4) of Ophiocordyceps sinensis and predict the antigenic sites. Methods The information of protein marker of O. sinensis was obtained by proteomics technology. The transcriptome database of O. sinensis and mycelium constructed in our lab were used to analyze IP4 unigenes and appropriate primers designed to amplify IP4, which was then cloned and sequenced. The conserved sequence of IP4, homology comparison, and the antigenic site were analyzed by DNAMAN6.0 and DNAStar software. Results Two alternative splice variants of IP4 were discovered from the transcriptome data and belonged to the invariant alternative splicing. The protein marker of O. sinensis was identified as cyanate hydratase, 465 bp, encoding 154 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in C-terminal of IP4. Analysis of DNAMAN 6.0 showed that species 1-39 aa and 55-81 aa were of higher species specificity, and DNAStar results showed that the epitope region of IP4 protein is distributed from 25 to 90 aa. Conclusion The optimal antigen region of IP4 lied in the region 25-39 and 55-81 aa, which lays a foundation for the subsequent large-scale preparation of IP4 protein and eutilizing ELISA to identify authenticity of O. sinensis.
ABSTRACT
Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit.
ABSTRACT
Objective Recent years, some studies have been studied on the biosynthesis of cordycepin, but it is not clear. To sequence the transcriptomes of the Ophiocordyceps sinensis which could provide the basis for revealing the bio-synthesis mechanism of cordycepin. Methods In this study, by Illumina/Solexa HiSeq 2500 technology, the transcriptomes of the O. sinensis fungus (anamorph) and the fruiting body (teleomorph) was sequenced, assembled and analyzed. By RT-PCR, the full lengths of RNRL (RNR large subunit) and RNRM (RNR small subunit) cDNA were cloned from the fresh O. sinensis fruit body. Results The pathway and the genes involved in cordycepin biosynthesis were predicted. Among of them, RNR was the critical enzyme in the metabolism of adenosine, also predicted to play an important role in the biosynthesis of cordycepin. From the transcriptome data, one large, one small subunits, and four similar sequences of RNR were found. RNRL mRNA was 2 733 bp, encoding 910 aa and RNRM mRNA 1 257 bp, encoding 418 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in RNRL, RNRM contained a ferritin-like conserved sequence. Conclusion This study would be established for revealing the bio-synthesis mechanism of cordycepin.
ABSTRACT
Natural Cordyceps sinensis(Dongchongxiacao) is an insect-fungi complex containing multiple Ophiocordyceps sinensis(≡Cordyceps sinensis) fungi and dead body of larva of the family of Hepialidae. But natural C. sinensis and O. sinensis fungi use the same Latin name, resulting in uncertainty of the specific meaning, even disturbing the formulation and implementation of governmental policies and regulations, and influencing consumer psychology onthe market. This paper reviews the history and current status of the indiscriminate use of the Latin name O. sinensis for both the natural insect-fungi complex C. sinensis and O. sinensis fungi and lists the rename suggetions. Some scholars suggested using the term O. sinensis for the fungi and renaming the natural C. sinensis "Chinese cordyceps". Others suggested renaming the natural C. sinensis "Ophiocordyceps & Hepialidae". Both suggestions have not reached general consensus due to various academic concerns. This paper also reviews the exacerbation of the academic uncertainties when forcing implementing the 2011 Amsterdam Declaration "One Fungus=One Name" under the academic debate. Joint efforts of mycological, zoological and botany-TCM taxonomists and properly initiating the dispute systems offered by International Mycology Association may solve the debate on the indiscriminate use of the Latin name O.sinensis for the natural insect-fungi complex,the teleomorph and anamorph(s) of O. sinensis fungi.
ABSTRACT
Isaria farinosa is the pathogen of the host of Ophiocordyceps sinensis. The present research has analyzed the progress on the molecular biology according to the bibliometrics, the sequences (including the gene sequences) of I. farinosa in the NCBI. The results indicated that different country had published different number of the papers, and had landed different kinds and different number of the sequences (including the gene sequences). China had published the most number of the papers, and had landed the most number of the sequences (including the gene sequences). America had landed the most numbers of the function genes. The main content about the pathogen study was focus on the biological controlling. The main content about the molecular study concentrated on the phylogenies classification. In recent years some protease genes and chitinase genes had been researched. With the increase of the effect on the healthy of O. sinensis, and the whole sequence and more and more pharmacological activities of I. farinosa being made known to the public, the study on the molecular biology of the I. farinosa would be deeper and wider.
ABSTRACT
Objective: To develop a rapid high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) method for the simultaneous determination of six polar compounds in Ophiocordyceps sinensis. Methods: A poroshell SB Aq column (50 mm × 4.6 mm, 2.7 μm) and gradient elution were used; The detection wavelength of compounds was set at 260 nm. The chromatographic peaks of the six investigated compounds in sample were identified by comparing their retention times with reference compounds. Results: All calibration curves showed good linearity (r > 0.999) within the tested ranges. The intra- and inter-day precisions of the six analytes were less than 0.8% and 2.1%, respectively, and the recoveries of the six analytes were between 95% and 103%. The validated method was successfully applied to the determination of six polar compounds in O. sinensis samples. Conclusion: The poroshell SB Aq column is suitable for the rapid analysis of polar components in Chinese materia medica on conventional HPLC system and the developed HPLC method is also helpful to the quality control of O. sinensis. © 2014 Tianjin Press of Chinese Herbal Medicines.
ABSTRACT
A HPLC-QTOF MS method was established for analysis of components in Ophiocordyceps sinensis . The HPLC analysis was performed on an Agilent Zorbax SB Aq (150 mmí4.6 mm, 5 μm) with gr adient elution (5 mmol·L-1 ammonium acetate aqueous solution-acetonitrile), flow rate was 0.8 mL·min-1 and detection wave-length was 260 nm. The developed method was successfully applied in analysis of three different samples in-cluding O. sinensis, Hirsutella sinensis ( anamorph of O.sinensis) and Cordyceps militaris. Nine compounds were i-dentified in both O.sinensis and H.sinensis, which eight compounds were identified in C.militaris.