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Objective @#To establish a microwave-assisted digestion-inductively coupled plasma mass spectrometry (ICP-MS) with an octopole reaction system for simultaneous determination of six heavy metals in peanuts, including Cr, Ni, As, Cd, Pb, Hg. @*Methods @#Peanut samples were shelled and crushed evenly, and 0.350 0 g was accurately weighed and digested with 5 mL nitric acid and 1 mL hydrogen peroxide in a digestion tank. Following microwave-assisted digestion, pure water was used to quantify the samples, and internal standards and an octopole reaction system were used to remove the interference. Then, the contents of six heavy metals were determined in peanuts by ICP-MS. The accuracy and precision of ICP-MS were evaluated using national criteria ( GBW 10013 and GBW 10044 ) and spike-and-recovery testing. @*Results @#The six heavy metals showed good linearity at the selected linear range ( r≥0.999 8 ). The detection limits of ICP-MS ranged from 0.001 4 to 0.023 8 ng/mL, and the spike-and-recovery rates ranged from 94.7% to 98.8%, with the relative standard deviations ranging from 0.7% to 3.6%. In addition, the determination results of the standard reference materials were all within the normal reference range. The detection of six heavy metals was 100.0% in 60 peanut samples, and the contents of six heavy metals were all low.@*Conclusion @#The established ICP-MS assay is feasible for simultaneous determination of multiple heavy metals in peanuts.
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Objective: To make a distinction between Ludisia discolor and its relatives genus in molecular level, SCoT markers were employed to assess the genetic relationship and construct the DNA fingerprint. Methods: Orthogonal design method were carried out to optimize the suitable SCoT-PCR reaction system based on five factors. The optimum annealing temperature and SCoT primers were also screened. The 12 germplasm resources were used as materials, the screened primers were selected to analyze the genetic relationship of 12 materials. POPGENE was used to calculate the genetic diversity, NTSYS was performed to analyze cluster, and DNA map was constructed. Results: The optimized SCoT-PCR reaction system was constructed and a total of 12 rich bands were screened out as the primers of SCoT molecular marker with polymorphism ratio of 98.98%. According to Nei's genetic similarity coefficient, a total of 12 materials were divided into three cluster when coefficient was 0.45. Goodyera schlechtendaliana was in category I with seven L. discolor lines, indicating that these samples had close relationship. In category II, there were three samples came from Anoectochilus roxburghii. Moreover, a green L. discolor sample was alone clustered into category III. The DNA fingerprint map by the SC8 primer could identify the 12 materials. Conclusion: There are rich genetic diversities in 12 samples of L. discolor and its relatives genus, and the construction of DNA fingerprint map provides a theoretical basis for the identification of L. discolor and its relatives genus, which were tested in this study.
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Objective To analyze the variation characteristics of rpoB,katG,inhA,rpsL and embB related genes of Mycobacterium tuberculosis(MTB)in Qinzhou,Guangxi.Methods PCR reverse point hybridization was used to detect 5 common resistance mutants of Mycobacterium tuberculosis in 237 MTB-DNA positive sputum samples.Results Among 237 cases of tuberculosis patients,72 cases(30.38%)were resistant to the four kinds of anti-TB drugs.The resistance mutation rate of rifampin,isoniazid and streptomycin was 2.53%, 13.92%,3.80%.The top 5 gene mutation detection loci of Mycobacterium tuberculosis were-15M,S531L and 43M.Conclusion The main drug-resistant strains are isoniazid resistance,and the mutation of inhA gene were the major one in Qinzhou,Guangxi.
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For rapid identification of Cervus nippon, C. elaphus and their hybridize samples, the specific PCR for mutual authentication of them was established based on the SNPs in COI and SRY sequence. C. nippon, C. elaphus and their hybridize samples were collected from different origins, total DNA of 24 identified samples were extracted, and the COI and SRY gene was seqenced. SNPs in the COI and SRY sequences of the samples were found by Clustul X 2.1 program. Primers for identifying C. nippon and C. elaphus were designed according to the SNP site, two multi-PCR reaction system were established to identify them. In addition, 24 samples which were randomly collected in different herbal medicine market were identified. The band special for C. nippon (232 bp)and band special for C. elaphus (518 bp) based on COI sequence,and the band special for C. nippon (803 bp)and band special for C. elaphus (425 bp) based on SRY sequence, were found using multi-PCR reaction, and three of the twenty-four samples were identified as the hybridize samples. The multi-PCR reaction system could be used to identify C. nippon, C. elaphus and their hybridize samples.
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Objective: For rapid identification of Houttuynia cordata and Gymnotheca chinensis, the specific PCR for mutual authentication of them was established based on the SNPs in matK sequence. Methods: H. cordata and G. chinensis samples from different origins were collected, total DNA of all samples was extracted, and the matK gene was seqenced. SNPs in the matK sequences of all the samples were found by ClustulX 2.1 program. Primers for identifying H. cordata and G. chinens were designed according to the SNP site, and specific PCR method was established to identify them, for rapid detection by addition of SYBR Green I dye. In addition, constructing a multi-PCR reaction system, and then the PCR reaction system was optimized. Results: The band special for H. cordata (185 bp) and band special for G. chinensis (389 bp) were found using specific PCR reaction and multi-PCR reaction, and SYBR Green I dye can be used for rapid detection. Conclusion: The multi-PCR reaction system could be used to identify H. cordata and G. chinensis.
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OBJECTIVE: To screen reaction system of the anticoagulation effect and establish reaction system of bioactivity assay of Hirudo in vitro and control the quality of Hirudo.
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OBJECTIVE: To establish an ICP-MS method for simultaneous determination of 19 trace elements, i.e. Na, Mg, Al, Si, K, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Ag, Cd, Sn, Hg, and Pb, in Andrographis Herba. METHODS: After microwave digestion treatment, samples were directly determined by inductively coupled plasma mass spectrometry based in octopole reaction system (ORS). RESULTS: The validated method indicated that the correlative coefficients (r) for all elements were above 0.9990. The limits of detections were in the range of 0.001-0.051 μg · kg-1. The reproducibility and stability were satisfactory with all RSDs below 10%. The spiked recoveries for Andrographis paniculata were between 88.64% and 102.98%. CONCLUSION: The method is simple, rapid and sensitive, which meets the requirement of trace analysis and can be used for the quality control of Andrographis Herba.
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Objective To establish and optimize AFLP reaction system used in providing necessary technique basis for genetic diversity analysis in Asarum sieboldii.Methods Leaves of A.sieboldii were used as experimental materials to analyze various essential elements of the whole process,such as quality of extracted DNA,time of restriction digest and ligation,concentration of Mg2+,primers and Taq DNA polymerase during PCR amplification etc.,so that the most optimal AFLP reaction system could be built up. Results The optimal AFLP reaction system of A.sieboldii has been constructed: in the genomic DNA extraction,mercaptoethanol being utilized and samples being incubated about 30 min at 65 ℃;the genomic DNA being digested by Trul Ⅰ and Pst Ⅰ for 3 h respectively;in PCR amplification,the final concentration of Mg2+ being 1.5 mmol/L and the volume of Taq DNA polymerase being 0.2 ?L.Conclusion The present reaction system for A.sieboldii is able to gain the favorable results of AFLP analysis and can be used in the genetic diversity research of the species.
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Objective To establish and optimize ISSR-PCR systems of Chrysanthemum morifolium and lay the foundation for its genetic diversity research. Methods Based on the analysis of variance, an orthogonal design was used to optimize the ISSR-PCR amplification system on C. morifolium by four factors (Taq polymerase, Mg2+, dNTP, and primer) at three concentration levels, respectively. Results A suitable ISSR reaction system was constructed with the 20 ?L reaction system containing 1.00 U Taq polymerase, 2.00 mmol/L Mg2+, 0.20 mmol/L dNTP, and 0.50 ?mol/L primer. Conclusion ISSR-PCR is significantly influenced by the concentration of Taq polymerase, Mg2+, and dNTP. This ISSR-PCR system could provide clear bands, reliable reaction system, and abundant polymorphisms . It is proved to be suitable for the study of the genetic diversity of C. morifolium
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Objective To study the genomic DNA extraction from Rhizoma Coptidus and optimization of RAPD reaction system. Methods Different methods, i.e. phenol method, CTAB method, low pH extraction medium with high salt, were used to genomic DNA extract from Rhizoma Coptidus. The DNA samples obtained by the above methods were tested by agarose gel electrophoresis and ultraviolet spectrometer. Results CTAB Method was considered to be an optimal technique. Based on the genomic DNA extracted by CTAB method, a reaction system suitable for Rhizoma Coptidus was established, that is, 25 ?L amplification reactions system containing 1?PCR buffer, 2 mmol/L Mg 2+, 100—150 ?mol/L dNTP, 20 ng primer, 40 ng template DNA, and 1 U Taq DNA polymerase. Conclusion CTAB Method and RAPD reaction system can be used to RAPD analysis in Rhizoma Coptidus.
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Objective Direct amplification of length polymorphism (DALP) as a new molecular marker was used to establish a set of stable DALP reaction system for the plants of Rhodiola L. Methods Some significant parameters of DALP reaction procedure were investigated and optimized by taking the DNA genome for the plants of Rhodiola L. as template. Results The reaction system was : 20 ?L reaction system containing 2. 5 mmol/L Mg2+ , 1. 25 mmol/L dNTPs, 60 ng DNA template, 1 ?L 5 pmol/L selective primer, 3 ?L 5 pmol/L reverse primer, selective primer: reverse primer is 1 : 3, and 2 U Taq DNA polymerase. Amplification program is 95℃ pre-denatured for 5 min, 94℃ denatured for 30 s, 50℃ annealed for 30 s, 72℃ extending for 1 min; after 30 cycles, and then 72℃ extending again for 10 min to the end of PCR reaction. Conclusion This DALP reaction system is efficient to identify the species and local populations for the plants of Rhodiola L. repeatedly with the stronger stability and reliability.