Realtime Fluorescent Quantitative PCR Analysis Of Biological Ingredient In Jinlong Capsule

Chinese Patent Medicines(CPM)is a compound preparation made of several and even ten tastes of chinese herbal medicines according to certain principles of healing formula.The composition of CPM is complex,production,sale,use,and many other aspects are not standardized,and quality standards are not specific and objective enough.In the driving of economic interests,some unscrupulous companies even replace some expensive medicines with chemical compositions,seeking to maximize benefits.Due to these,poor treatment effect,or even serious adverse reactions,are also problems which are necessary to confront in CPM's development path.Therefore,the accurate identification and quantitative analysis of each component of the medicine-based sources,is the primary task to ensure drug safety and clinical efficacy.Traditional methods for the identification of Chinese medicine mainly include microscopic identification,and other physical and chemical identification.In recent years,a growing number of new methods and instruments have been applied to the area of medicine identification,such as chromatographic,spectroscopic identification and so on.In addition,with the rapid development of molecular biology and related technologies,nucleic acid sequence analysis of species based identification techniques have been springing up rapidly.Real-time fluorescent quantitative PCR(Q-PCR),based on the conventional PCR technology,adds the fluorophore to the reaction system.It determines the initial concentration of template DNA by collecting the fluorescence signal generated in response to the realization of the entire PCR process monitoring,and finally by analyzing the threshold cycle number(Threshold cycle,Ct value)and the standard curve.It has the specificity of nucleic acid hybridization techniques,sensitivity and simple operability of conventional PCR techniques,and accuracy of the characteristic spectral technique.In this paper,we discussed the feasibility of the application of Q-PCR technology in the identification of Jinlong Capsule.Jinlong capsule is an anti-cancer drug consisted of fresh bodies of Gekko sp.,Deinagkistrodon acutus,and infant Bungarus multicinctus in a ratio of 2:1:1.Freezing,thawing,and filtration are used in its process of production,which reserves the genetic material to the hilt,and provides a good foundation for the experiment.The establishment of Q-PCR identification method forJinqian Baihua She(Bungarus Parvus,JBS)1 PurposeTo identify and quantify JBS from Jinlong capsule with fluorescent quantitative PCR by designing the specific primers of JBS based on COI sequence.2 Method2.1 Samples collectionA total of 15 specimens from 11 snake species(including B.multicinctus and its adulterants)were obtained from various locations in Guangdong Province,Hunan Province,Jiangxi Province and Guangxi Zhuang Autonomous Region in China.2.2 Species-specific primer designBased on primer designing principles,species-specific primer of JBS was designed.2.3 DNA extractionThe total DNA was extracted using DNA kit following the manufacturer's instruction.2.4 COI Barcode amplificationThe universal primers were used to amplify the COI barcode region.PCR was amplified in a total reaction volume of 10?l containing 0.6?l genomic DNA,0.3?l each primer,2×Taq Plus PCR MasterMix 5?1,with 3.8?l ddH2O.Thermal cycling was performed with an initial denaturing at 93 ? for 5min and annealing at 55 ? for 2min,followed by 35 cycles of 93? for 30s,55? for 45s,and 70 ? for 30s,with a final extension at 70 ? for 5min and chilling to 4?.After PCR amplification,PCR products were detected by agarose gel electrophoresis.2.5 Diagnostic PCR of JBSDNA of JBS and its adulterants were amplified using species-specific primers,and a negative control without DNA template was set.PCR reaction system was described in step 2.4.Thermal cycling was performed with an initial denaturing at 94 ? for 5min and annealing at 50?for 2min,followed by 30 cycles of 94? for 30s,50? for 50s,and 72? for 30s,with a final extension at 72? for 5min and chilling to 4?.The DNAs were fractionated by agarose gel electrophoresis and visualized by EB staining under UV.As described above,the annealing temperature,primer concentration and the number of cycles were carried out to explore and optimize the PCR reaction conditions respectively.2.6 Q-PCR identification for JBSAccording to the experimental conditions of PCR explored in advance,JBS and its adulterants were identified by Q-PCR.A negative control without DNA template was set.PCR was amplified in a total reaction volume of 20?l containing 1.2?l genomic DNA,0.6?l each primer,SYBR(?)Premix Ex TaqTM 10?l with 7.6?l ddH2O.Reactions were performed with 30s of pre-incubation at 94? followed by 25 amplification cycles of 30s at 94?,50s at 65? and 30s at 72?(with single fluorescence acquisition).After that,melting curve analysis(MCA)was further performed to confirm the expected PCR products as follows:after PCR,samples were denatured for 5s at 95 ? followed by lmin at 60? and 0s at 95 ?(with continuous fluorescence acquisition),and chilling to 4?.Amplification curves,melting curves,threshold cycler(Ct)value,Tm value and other related data were recorded.2.7 Establishment of quantitative method for JBS2.7.1 Standard curve establishmentAmplification of the COI fragment of JBS was similar to that described in section 2.4,except for that the reaction volume was expanded to 50?l.PCR products were purified using Universal DNA Puriication Kit following the manufacturer's instructions.The concentration and the number of copies of purified PCR products were determined.The COI fragment of JBS was diluted 10 times,then the diluted products instead of DNA template for Q-PCR were carried out with the method of step 2.6.Amplification curves,melting curves,threshold cycler(Ct)value,Tm value and other related data were recorded.Choosing different dilution ratio samples amplified normally,standard curve was established by linear regression analysis in which the number of copies of samples'logarithm(base number is 10)was X axle,corresponding Ct was Y axle.2.7.2 Verification of quantitative method for JBSAccording to the proportion of prescriptions and manufacturing method of Jinlong capsule,four groups experiments(all quality goods,quality goods/adulterants 1:1,quality goods/adulterants 1:4,all adulterants)were established.Parellel experiments were set in first three groups.DNA extraction of the self-made samples same to the step 2.3.Using genomic DNA from self-made samples as templates,Q-PCR were performed samed to the method of step 2.6.Each sample was repeated once,and a negative control without DNA template was set.Amplification curves,melting curves,threshold cycler(Ct)value,Tm value and other related data were recorded.3 Results3.1 Species-specific primer designA pair of species-specific primer(COI37/COI337)for identification of JBS was designed.COI37:5,-AATCG GAGCC TGTCT AAG-3';COI337:5'-GACTG TTCAACCTGT GCC-3'.3.2 COI Barcode amplificationAfter electrophoresis,the COI fragments amplified products of JBS and its adulterants both got amplified band of about 700bp length.While no amplification product was obtained for negative control.3.3 Diagnostic PCR of JBSDiagnostic PCR conditions include an initial step at 94 ? for 5min,50 ? for 2min,and 25 cycles of 94? for 30s,65? for 50s,and 72? for 30s,with a final extension at 72 ? for 5min.Under the optimized conditions,a single,distinct,and brightly resolved band of 300 bp was obtained only for JBS,while no amplification product was obtained for the others.3.4 Q-PCR identification for JBSUsing the primer pair COI37/COI337,only JBS appeared amplification curves and melting curves in the Q-PCR reaction,while no amplification product was obtained for other samples and negative control.3.5 Establishment of quantitative method method for JBSUsing the above method,the standard curve for quantitative analysis of JBS was established.The standard curve equation(y =-2.9247x + 31.725,y is Ct value,x is LgN,N is the copies of COI fragment from JBS).Samples from H1 to H9(obtained B.multicinctus)appeared amplification curves and melting curves in the Q-PCR reaction,while no amplification product was obtained for HiO(no B.multicinctus)and negative control.The number of copies of COI of JBS was respectively 5.722×106 copies/?l,2.397×106copies/?l and 1.138×106copies/?1 in all quality goods,quality goods/adulterants 1:1,quality goods/adulterants 1:4.The results accorded with proportion of adding amount roughly.The establishment of Q-PCR identification method forShougong(Gekko chinensis)1 PurposeCOI barcode-based diagnostic PCR system integrated with fluorescent quantitative PCR,to identify and quantify Gekko chinensis from Jinlong capsule.2 MethodSame as "The establishment of Q-PCR identification method for Jinqian Baihua She(Bungarus Parvus,JBS)" narrated above.3 Results3.1 Species-specific primer designA pair of species-specific primer(GZG1/GZG2)for identification of Gekko chinensis were designed.GZG1:5'-GCAAC TGACT AATCC CACT-3';GZG2:5'-TGCTT CTTCT GGCGT AG-3'.3.2 COI Barcode amplificationAfter electrophoresis,the COI fragments amplified products of Gekko chinensis and its adulterants both got amplified band of about 700bp length.While no amplification product was obtained for negative control.3.3 Dagnostic PCR of Gekko chinensisDiagnostic PCR conditions include an initial step at 94 ? for 3min;and 25 cycles of 94? for 30s,57? for 45s,and 72? for 30s,with a final extension at 72?for 5min.Under the optimized conditions,a single,distinct,and brightly resolved band of 150 bp was obtained only for Gekko chinensis,while no amplification product was obtained for the others.3.4 Q-PCR identification for Gekko chinensisUsing the primer pair GZG1/GZG2,only Gekko chinensis appeared amplification curves and melting curves in the Q-PCR reaction,while no amplification product was obtained for other samples and negative control.3.5 Establishment of quantitative method method for Gekko chinensisUsing the above method,the standard curve for quantitative analysis of Gekko chinensis was established.The standard curve equation(y =-3.0127x + 34.501,y is Ct value,x is LgN,N is the copies of COI fragment from Gekko chinensis).Samples from H1 to H9(obtained Gekko chinensis)appeared amplification curves and melting curves in the Q-PCR reaction,while no amplification product was obtained for H10(no Gekko chinensis)and negative control.The number of copies of COI of Gekko chinensis was respectively 11.511×106copies/?l,6.416×106copies/?l and 2.553×106 copies/?l in all quality goods,quality goods/adulterants 1:1,quality goods/adulterants1:4.The results accorded with proportion of adding amount roughly.The establishment of Q-PCR identification method forAgkistrodon(Deinagkistrodon acutus)1 PurposeCombining the species-specific primers to identify Agkistrodon recorded on the Chinese Pharmacopoeia with Q-PCR,to identify and quantify Agkistrodon from Jinlong capsule.2 MethodSame as "The establishment of Q-PCR identification method for Jinqian Baihua She(Bungarus Parvus,JBS)" narrated above.3 Results3.1 COI Barcode amplificationAfter electrophoresis,the COI fragments amplified products of Deinagkistrodon acutus and its adulterants both got amplified band of about 700bp length.While no amplification product was obtained for negative control.3.2 Dagnostic PCR of Deinagkistrodon acutusDiagnostic PCR conditions include an initial step at 95 ? for 30s,25 cycles of 95 for 30s,50? for lmin,and 72? for 30s,with a final extension at 72? for 5min.Under the optimized conditions,a single,distinct,and brightly resolved band of 300 bp was obtained only for Deinagkistrodon acutus,while no amplification product was obtained for the others.3.3 Q-PCR identification for Deinagkistrodon acutusUsing the primer pair DK1/DK2,only Deinagkistrodon acutus appeared amplification curves and melting curves in the Q-PCR reaction,while no amplification product was obtained for other samples and negative control.3.4 Establishment of quantitative method method for Deinagkistrodon acutusUsing the above method,the standard curve for quantitative analysis of Deinagkistrodon acutus was established.The standard curve equation(y =-3.2617x +47.014,y is Ct value,x is LgN,N is the copies of Cytb fragment from Deinagkistrodon acutus).Samples from H1 to H9(obtained Deinagkistrodon acutus)appeared amplification curves and melting curves in the Q-PCR reaction,while no amplification product was obtained for H10(no Deinagkistrodon acutus)and negative control.The number of copies of COI of Deinagkistrodon acutus was respectively 7.056x109copies/?l,4.166×109copies/?l and 1.668×109copies/?l in all quality goods,quality goods/adulterants 1:1,quality goods/adulterants 1:4.The results accorded with proportion of adding amount roughly.ConclusionsWe for the first time established the species-specific Q-PCR identification method for JBS,Gekko chinensis and Agkistrodon.This method has good specificity,high sensitivity.It not only can be used for the qualitative identification of self-made samples,but also can be used for the quantitative analysis.