Identification Of Adulteration In Fritillariae Cirrhosae Bulbus Based On MLPA-HRM Assay

Fritillariae cirrhosae bulbus,a multi-origin plant derived from Fritillaria cirrhosa,F.przewalskii,F.delavayi,F.unibracteata,and F.unibracteata var.wabuensis,is an important traditional Chinese medicine in China,which has remarkable effects on clearing away heat and moistening the lungs,relieving cough and eliminating phlegm,and swelling.Known as the holy medicine for moistening the lungs,it has good curative effects and fewer side effects than other congeneric medicine.However,due to long growth cycle,the shortage of resources,growing market demand and valued medical use,it is difficult to meet clinical demands.As a result,the problem of adulteration is becoming more and more serious,which seriously damages the curative effect and medication safety of F.cirrhosae bulbus.Therefore,it is very important to establish a rapid,convenient,and operable method to identify F.cirrhosae bulbus and avoid confusion,which is of great significance to the regulation of Chinese medicinal materials market and the healthy development of traditional Chinese medicine.At present,there are many methods to identify Fritillaria,but most of them are cumbersome,time-consuming,unable to identify mixed samples,or ambiguous and have the risk of pollution.In this study,combined with high-resolution melting curve assay,the multiplex ligation probe amplification(MLPA)was employed for its advantages of high efficiency,specificity,and sensitivity.F.cirrhosae bulbus and its common adulterants including F.thunbergii bulbus,F.hupehensis bulbus,F.ussuriensis bulbus and F.pallidiflorae bulbus were taken as research objects,and pairs of MLPA probe based on ITS1 sequence were designed respectively.After denaturation,hybridization,ligation,and melting,the results were present according to the high-resolution melting curve.In this study,five reference samples of Fritillaria were analyzed to detect the specificity,sensitivity,and reproducibility of MLPA-HRM assay,and the mixed samples of F.cirrhosae bulbus and F.pallidiflorae bulbus with different proportions were detected.The results show that MLPA-HRM assay can detect Fritillariae cirrhosae bulbus,F.thunbergii bulbus,F.hupehensis bulbus,F.ussuriensis bulbus and F.pallidiflorae bulbus simultaneously;the probe designed in this study has strong specificity,single probe and mixed probe can only hybridize with the corresponding species in a system to produce positive signals of the specific species,and no non-specific peak was obtained with any probes;the sensitivity is high,the detection limit of MLPA-HRM for Fritillariae cirrhosae bulbus was 0.19 ng at the DNA level,and the detection limit of MLPA-HRM for F.pallidiflorae bulbus was 1.3ng at the DNA level;the repeatability is good.Theoretically,the reproducibility of the results depends on the purity of DNA samples and the thoroughness of mixing reaction systems,which can be generally guaranteed.Furthermore,this method can detect mixed samples.When the content of F.pallidiflorae bulbus is 10% or higher,the melting curve will show double peak which belongs to Fritillariae cirrhosae bulbus and F.pallidiflorae bulbus.Seventy Fritillaria materials collected from various herbal medicine markets were analyzed using the MLPA-HRM assay,and to validate and confirm the MLPA-HRM results,all of them were also analyzed in parallel with the conventional method(PCR-RFLP)according to Pharmacopoeia 2015.The results of MLPA-HRM assay showed that 43 F.cirrhosae bulbus samples were found,18 non-F.cirrhosae bulbus samples were found,and nine mixed samples were found.However,with the PCR-RFLP method,52 samples were F.cirrhosae bulbus,and 18 samples were not F.cirrhosae bulbus.Thus,PCR-RFLP could not identify if the F.cirrhosae bulbus sample was mixed with adulterants,the MLPA-HRM assay could just make up for this deficiency.To further expand the scope of application of MLPA-HRM assay,this method has been successfully applied to the detection of sixteen Chinese Fritillaria patent medicines purchased from various pharmacies,and other medicinal materials have no interference with the experimental results.The results showed that among the 11 batches of Shedan Chuanbei powder,nine F.cirrhosae bulbus samples were found,two mixed samples were found;the four batches of compound Fritillaria tablets were not F.cirrhosae bulbus;among one batches of compound fritillary ammonium chloride tablets,F.cirrhosae bulbus was found.Similar to the detection results of Fritillaria materials,the PCR-RFLP method could not identify if the F.cirrhosae bulbus sample was mixed with adulterants,and other results were consistent.Compared with PCR-RFLP method,the method established in this study can be used for rapid detection of Chinese patent medicines,and is more effective and accurate monitoring of drug safety.In conclusion,MLPA-HRM assay can be used to control the adulteration of F.cirrhosae bulbus in the market and the original powder of Fritillaria in Chinese patent medicines.In addition,by improving this method,it can also be used as a prototype to detect other species.