In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
DNA Primers ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; chemistry ; standards ; Fluorescent Dyes ; chemistry ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; instrumentation ; methods ; Quality Control ; Staining and Labeling

To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully.
Alleles ; Cistanche ; classification ; genetics ; DNA Primers ; genetics ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Drug Contamination ; prevention & control ; Phylogeny ; Polymerase Chain Reaction ; methods

The sequences of ITS, matK, rbcL and psbA-trnH of 9 Gynostemma species or variety including 38 samples were compared and analyzed by molecular phylogeny method. Hemsleya macrosperma was designated as outgroup. The MP and NJ phylogenetic tree of Gynostemma was built based on ITS sequence, the results of PAUP phylogenetic analysis showed the following results: (1) The eight individuals of G. pentaphyllum var. pentaphyllum were not supported as monophyletic in the strict consensus trees and NJ trees. (2) It is suspected whether G. longipes and G. laxum should be classified as the independent species. (3)The classification of subgenus units of Gynostemma plants is supported.
Gynostemma ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Sequence Analysis, DNA

Objective:To investigate the changes of the vertical height and width of the alveolar bone six months after the alveolar ridge preservation in periodontal compromised molar sites of severe alveolar bone defects with clinical direct measurement,parallel periapical radiographs,and cone-beam computed tomography (CBCT),and to analyze the effect of the three different methods of measurement.Methods:In this study,20 subjects requiring tooth extraction on account of periodontal disease with a total of 23 ex-tracted molars were enrolled.Extractions were performed atraumatically and patients were received alveo-lar ridge preservation procedure with Bio-Ossand Bio-Gide.Clinical direct measurements were taken after tooth extraction and during the implant surgery 6 months later,CBCT scans and parallel periapical radiographs were taken immediately after ridge preservation and 6 months later.The changes of alveolar ridge width and vertical height after six months were measured and analyzed through the above-mentioned three methods and the similarities and differences of the measured effect were compared.Results:There were no significant difference of alveolar vertical height in the center of the extraction sites,the center of distal aspect,and distobuccal aspect between the clinical direct measurements and the CBCT measure-ments (P>0.05),alveolar vertical height in other points and alveolar width measurements were statical-ly significant (P<0.05).After 6 months,1 0 sites of 1 0 subjects were received a flap and re-entered to perform dental implants surgery.The vertical height in the center of alveolar increased significantly and the changes of alveolar vertical height of clinical direct and CBCT measurement were (6.1 5 ±1 .73)mm and (6.59 ±2.53)mm,respectively.The measurements of the width of the alveolar bone were (8.45 ± 1 .1 8)mm and (8.52 ±1 .27)mm,respectively.The measurements of the two methods were not statisti-
cally significant (P>0.05).The change of the alveolar height in the center of the extraction socket after six months measured by parallel periapical was (5.84 ±4.28)mm,which was closed to the clinical di-rect measurement and the CBCT measurement.Conclusion:Clinical direct measurement and CBCT measurement were largely consistent in the evaluation of the alveolar bone height and width after the alveolar ridge preservation using deproteinized boving bone mineral (DBBM,Bio-Oss)and bioabsor-bable collagen membrane (Bio-Gide)in periodontal compromised molar sites of severe bone defects.

To explore a new method for identification of Mongolian patent medicine (MPM) by PCR amplification of specific alleles. Eight kinds of MPM were used to study the identification of "Digeda" raw materials. The total DNA of Lomatogonium rotatum and Corydalis bungeana samples were extracted through modified CTAB method, psbA-trnH sequence was amplified by PCR and sequenced directionally. Specific primer was designed. The DNA of 8 kinds of MPM also was extracted and purified by the commercial DNA purification kits. The rbcL and two pair of specific primers sequences were amplified. The specific amplified products were sequenced in forward directions. All specific sequences were aligned and were analyzed. The results indicated that L rotatum can be identified by specific primers from Digeda-4 Tang, Digeda-8 San, Digeda-4 San, and C. bungeana medicinal materials can be identified by specific primers from Li Dan Ba Wei San, Yi He Ha Ri-12 and A Ga Ri-35. PCR amplification of specific alleles can stably and accurately distinguish raw medicinal materials in MPM.
Alleles ; DNA Primers ; genetics ; DNA, Plant ; genetics ; Medicine, Mongolian Traditional ; Molecular Sequence Data ; Plants, Medicinal ; classification ; genetics ; Polymerase Chain Reaction ; methods

BACKGROUND: It is found reported that polymorphism of Fok 1 restriction endonuclease cut site on exon 2 of 5' end start codon of 5' end start codon (SC), which affected the structure of VDR amino acids,and was relative related to bone mineral density(BMD).OBJECTIVE: To analyze the association between Vitamin D receptor gene (Fok 1) polymorphisms and osteoporosis in the elderly men.DESIGN: case-controlled trialstudy.SETTING: Institute of Gerontology, Chinese PLA General Hospital and Department of Endocrinology,Second Artillery General Hospital of Chinese PLA.PARTICIPANTS: A total of 26 elderly men with osteoporosis at out-patients clinic of Chinese PLA General Hospital and Department of Endocrinology,Second Artillery General Hospital of Chinese PLA from January 2002 to June 2002 were selected involved as osteoporosis case group,with and the average age of was (70±5) years, and BMD in osteoporosis group was 2.0-2.5 SD lower than 2.0-2.5 SD of the peak of BMD. Totally 66 healthy men with average age of (70±5)years were selected as control group during at the same time. All the subjects signed the informed consent,who were Beijing inhabitants of Han nationality, and there was no blood relationship among them.METHODS:VDR-Fok1 genotypes in both groups were detected with by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP),and distributiondistribution of VDR-Fok 1 genotypes were analyzedanalyzed.MAIN OUTCOME MEASURES: distribution Distribution of VDR-Fok1genotypes in both each groups.RESULTS: Totally 66 healthy elderly men and 26 elderly men with osteoporosis entered analysis of results. The frequencies of FF, Ff and ff genotype were found to be 42%, 42% and 15% in control group, and 15%,50%,35% in osteoporosis group, respectively,and there was significantly different between two groups(x2=12.078,P ＜ 0.01).Frequency of allele were significantly different between control group and osteoporosis group (64%,36% vs 40%,60%, x2=8.232,P ＜ 0.01).CONCLUSION: There is a significant difference in the frequency distrinution of VDR gene start codon polymorphism between healthy elderly men and those with osteoporosis.

Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.
Base Sequence ; Chloroplasts ; genetics ; Cluster Analysis ; DNA Barcoding, Taxonomic ; DNA, Chloroplast ; genetics ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Drugs, Chinese Herbal ; chemistry ; Forsythia ; chemistry ; genetics ; Phylogeny ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; Ribulose-Bisphosphate Carboxylase ; genetics ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVETo analyze the forces of rotational wall vessel (RWV) bioreactor on small tissue pieces or microcarrier particles and to determine the tracks of microcarrier particles in RWV bioreactor.

METHODSThe motion of the microcarrier in the rotating wall vessel (RWV) bioreactor with both the inner and outer cylinders rotating was modeled by numerical simulation.

RESULTSThe continuous trajectory of microcarrier particles, including the possible collision with the wall was obtained. An expression between the minimum rotational speed difference of the inner and outer cylinders and the microcarrier particle or aggregate radius could avoid collisions with either wall. The range of microcarrier radius or tissue size, which could be safely cultured in the RWV bioreactor, in terms of shear stress level, was determined.

CONCLUSIONThe model works well in describing the trajectory of a heavier microcarrier particle in rotating wall vessel.

Bioreactors ; Computer Simulation ; Microspheres ; Motion ; Porosity ; Rheology ; Rotation ; Stress, Mechanical ; Tissue Engineering ; methods

To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles, 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted, trnL-trnF sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature, dNTP, etc were optimized. All samples were amplified by PCR with specific primer, DNA from Astragali Radix would be amplified 136 bp, whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix.
Alleles ; Astragalus Plant ; classification ; genetics ; DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; Polymerase Chain Reaction

To evaluate the effect of PCR enhancer on molecular identification of Chinese herbal medicine, and select the optimal enhancers suitable for traditional Chinese medicine (TCM), genomic DNA from 180 kinds of Chinese herbal medicine was extract by CTAB method and alkaline lysis method, respectively. PCR success rate of five universal fragments (ITS2, psbA-trnH, rbcL, matK, trnL-trnF) was compared in a PCR system with and without enhancer. PCR efficiency of Real-time PCR was also compared in a PCR system with and without enhancer. Results showed that PCR success rate of ITS2,psbA-trnH, rbcL fragment was increased by using PVP and BSA. The PCR efficiency was decreased by PCR enhancer in Real-time PCR system. The results indicate that BSA and PVP as PCR enhancer can dramatically increase PCR success rate and genotyping accuracy in TCM molecular authentication.
DNA, Plant ; analysis ; genetics ; Drugs, Chinese Herbal ; Genotype ; Polymerase Chain Reaction ; methods ; Quality Control ; Temperature