Study on the Quality Standard of Qiqilian Capsule / 中国药房

OBJECTIVE:To establish the quality standards for Qiqilian capsule. METHODS:TLC was used to identify the As-tragali Radix,Phellodendri chinensis,Coptidis rhizom. HPLC was used to determine the contents of ginsenosides Rg1,ginsenosides Rb1 and notoginsenosides R1. The column was Shim-pack VP-ODS C18 with mobile phase of acetonitrile-water(gradient elution) at flow rate of 1.0 ml/min,detection wavelength was 203 nm,column temperature was 20 ℃. RESULTS:TLC of Astragali Radix,P. chinensis,C. rhizom showed cleer sports and good separation. The linear range was 0.9-9.0 μg for ginsenosides Rg1,0.94-9.4 μg for ginsenosides Rb1 and 0.3-3.0 μg for notoginsenosides R1(r≥0.999 5);RSDs of precision,reproducibility and stability test were lower than 3.0%;recoveries were 96.08%-99.75%(RSD=1.52,n=6),97.03%-99.75%(RSD=1.10,n=6)and 96.38%-98.55%(RSD=0.90,n=6),respectively. CONCLUSIONS:The method is simple,good reproducibility,and can be used for the quality control of Qiqilian capsule.

Prokryotic expression of CFP-10 protein of Mycobacterium tuberculosis / 中国医师杂志

Objective To construct recombinant plasmid containing CFP-10 gene of Mycobacterium tuberculosis(MTB).Methods The gene fragment of CFP-10 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pET-32a(+) vector.The recombinant plasmid pET-32a-CFP-10 was transformed into E.coli BL21 (DE3) and induced by IPTG.Results CFP-10 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and thepET-32a(+) prokaryotic recombinant plasmid was constructed successfully.The recombinant protein was expressed with the induction of IPTG.Conclusions The prokaryotic expression vector for CFP-10 was successfully constructed and the recombinant protein was highly expressed in E.coli BL21 (DE3),which lays a foundation for its subsequent immunological function study.

Development of a diagnosis model for active pulmonary tuberculosis using mass spectrometry and pro-tein chip / 中华微生物学和免疫学杂志

Objective To develop a diagnosis model for active pulmonary tuberculosis. Methods The proteomic fingerprinting of 264 sera from active tuberculosis patients and controls were analyzed using the surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and protein-chip technology. The peaks were detected and filtrated by Ciphergen PrnteinChip(R) Software (version 3.1.1). Using the Biomarker Pattern 5.0 software, a diagnostic model was developed for diagnosis of active tuberculosis. Re-sults Fifty protein peaks were significantly different between the patients with active pulmonary tuberculosis and the controls with overlapping clinical features (P<0.01). Five protein peaks at 4360, 3311, 8160, 5723, 15173 m/z were chosen for the system classifier and the development of diagnosis model 1. The model differenti-ated the patients with active pulmonary tuberculosis from the controls with a sensitivity of 83.0%, and a speci-ficity of 89.6%. The diagnostic accuracy was up to 86.4%. Three protein peaks at 5643, 4486, 4360 m/z were chosen for the system classifier and the development of diagnosis model 2. The model differentiated the pa-tients with active pulmonary tuberculosis from the controls with a sensitivity of 96.9%, and a specificity of 97.8%. The diagnostic accuracy was up to 97.3%. Conclusion It might be a new diagnostic test for the de-tection of sera from the patients with active pulmonary tuberculosis using SELDI-TOF-MS and protein chip.

Relationship of inflammatory factors in blood serum and immune status of patients with active pulmonary tuberculosis / 第三军医大学学报

Objective To investigate the changes of inflammatory factors in the blood serum and their relationship with the immune status of patients with active pulmonary tuberculosis(TB).Methods A total of 97 cases of pulmonary tuberculosis were included from Feb 2003 to Oct 2005,57 of active TB,40 in resting period.Another 41 healthy people were used as normal control.ELISA and APAAP method were used to detect the level of TNF-?, IL-1,IL-6 and the changes of CD_(4),CD_(8)and CD_(4)/CD_(8).Results The levels of IL-1,IL-6,TNF-? were(15.3?1.3),(80.5?7.3) and(77.2?9.8) ng/ml in the normal controls,(33.7?3.6),(293.6?30.5) and(190.7?25.2) in the patients of active TB,and(18.2?2.1),(130.7?14.5),(87.5?10.2) ng/ml in the patients at resting period,which were highest in the patients of active TB.The ratio of CD_(4)and CD_(4)/CD_(8) was(32.3?2.9)% and(0.83?0.17) in the patients of active TB,lower than(48.2?4.4)% and(0.83?0.17) of normal controls.Conclusion The increase of inflammatory factors and decrease of immune activity were the clinical characteristic of patients with active pulmonary tuberculosis,which are of inverse relationship.

embB mutations in Mycobacterium tuberculosis ethambutol-resistant isolates / 中华检验医学杂志

Objectives To understand the mutations of embB genes in M. tuberculosis isolates, and to evaluate their clinical value. Method 102 clinical isolates were identified for their mycobacterial species, and then analyzed their embB genes with PCR SSCP, PCR RFLP, and PCR direct sequencing. Results Mycobacterium tuberculosis strain H 37 R v was used as a control. 102 clinical isolates all had the same 16S rDNA SSCP profiles as M. tuberculosis . Forty one drug sensitive isolates had normal embB SSCP and RFLP profiles. Of 61 ethambutol resistant isolates, 23 (37.7%) displayed abnormal embB SSCP profiles. Eight isolates had abnormal RFLP profiles. All embB mutations situated at codon 306, whose EMB MICs were more than 20 ?g/ml. Eight isolates had ATG to ATA or ATT mutations at codon 306. Thirty isolates had ATG to GTG or CTG mutations at codon 306, whose EMB MICs were more than 30 ?g/ml. Conclusions Ethabutol resistances in some M. tuberculosis isolates were due to mutations on embB genes. PCR SSCP and PCR RFLP method might become a simple and rapid diagnostic test for genotypes of M. tuberculosis ethabutol resistance.

Rapid Detection of Mycobacterium Tuberculosis Streptomycin-resistant Genes by PCR-reverse Dot Blot Hybridization / 中国医师杂志

Objective To study the rapid detection of mycobacterium tuberculosis resistance to streptomycin by reverse dot blot hybridization technique. Methods The oligonucleotide probes of streptomycin-resistant genes (rpsL and rrs) were prepared and dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with the oligonucleotide probes on the membranes. PCR-SSCP and PCR-direct sequencing (PCR-DS) techniques were used to detect the target fragment of M.tuberculosis as control. Results In 53 M. tuberculosis clinical isolates, the consistent rate of three detection methods was 100%. Both the SSCP mapping of rpsL and rrs genes and the results of membrane hybridization in 9 drug-sensitive strains were identical to those in M. tuberculosis standard strain H37Rv. Of 44 streptomycin-resistant strains, 33 strains had AAG→AGG mutation at the codon 43 of rpsL gene, 6 strains had A→C mutation at the 513 site of rrs gene, 1 strain had A→T mutation at the 513 site of rrs gene, and the detection rate of the target genes mutation was 90 9%. In 53 M.tuberculosis clinical isolates, 40 resistant strains and 9 sensitive strains to streptomycin could be detected using dot blot hybridization and the consistent rate with the in vitro susceptibility test was 92 6%(49/53). Conclusion The reverse dot blot hybridization technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It possessed the simple and rapid characteristics, and could be used to detecte streptomycin-resistant M.tuberculosis clinical strains.