ABSTRACT
Objective To investigate the application and significance of the MS/MS method and the HPLC method for the screening and diagnosis of genetic metabolic disease phenylketonuria (PKU) .Methods The MS/MS method and the HPLC method were adopted to analyze the concentrations of phenylalanine(Phe) and tyrosine(Tyr) and its ratio in the dried blood spot specimen on filter paper and the whole blood specimen in 1 860 children aged from 3 d to 11 years old .Results The linear ranges of Phe by the MS/MS method and the HPLC method were 26 .02 - 101 .11 μmol/L and 32 .04 - 132 .50 μmol/L ,which of Tyr were 41 .50 -253 .07 μmol/L and 32 .85 - 111 .50 μmol/L ,the average recovery rates of Phe were 97 .36% and 98 .43% ,which of Tyr were 96 .71% and 98 .99% ,in‐run CV of Phe were 4 .31% and 3 .97% ,which of Tyr were 4 .09% and 4 .01% ,between‐run CV of Phe were 5 .73% and 4 .58% ,which of Tyr were 6 .01% and 5 .24% ,respectively .Conclusion Both methods can sensitively and specif‐ically detect blood Phe and Tyr concentrations and meet the requirements of screening and diagnosis of PKU .
ABSTRACT
0bjective To investigate the serum total bilirubin level of healthy population in northwest area of China.Methods According to the guiding principle and the screening standard of C28-P3,722 individuals conforming to the screening standard from October 2011 to January 2012 were collected.Serum total bilirubin was determined with diazo reagent method(Roche)on the P module of Hitachi 7600 automatic biochemistry analyzer,and the vanadate reagent method(WAKO)on the D module of the Hitachi 7600 automatic biochemistry analyzer,respectively.The detection results were analyzed between different analysis systems,between country and city and among different age groups by SPSS13.0.The top and bottom limitations of 95% reference interval recommen-ded by the C28-P3 file were calculated by the non-parametric method.Results Serum total bilirubin detection results had no statis-tically significant differences between the two kinds of detection methods,between rural and urban populations,among all ages and genders (P >0.05).The ultimate reference interval of serum total bilirubin in the northwest area was 2.19 -29.29 μmol/L.Con-clusion The differences of serum total bilirubin reference interval exist between the healthy population in the Northwest area of China and the current reference interval used in domestic and foreign population.Establishing the reference intervals of new bio-chemical test item suitable for China′s population will provide the scientific basis for the evaluation of disease diagnosis,treatment, prognosis judgment and health assessment in the Chinese population.
ABSTRACT
Objective To study the reference interval of serum iron and magnesium of healthy people in northwest region . Methods 722 healthy people aged 18- 0 .05) ,and the re-sults was combined into one group .Difference of iron test results between male and female was statistically significant (P<0 .05) , and grouping was performed according to gender ,that was ,male:10 .05-36 .21 mmol/L ,female:6 .19-30 .87 mmol/L .Difference of magnesium test results between male and female showed no statistical significance (P<0 .05) ,and was combined into one group , with the reference interval of 0 .74-1 .06 mmol/L .Conclusion The iron and magnesium 95% reference intervals of healthy people in northwest region are narrower than those of the National test results .
ABSTRACT
Laboratory diagnostics is one of the most fast developing medical sciences.But the teaching quality falls behind due to the traditional education model.Based on the concept of ‘three-stage fusion' of the subject and ‘five abilities' of the training objects,‘three-highlight & stereo' teaching style was established and put through the course,which means to emphasize function,practice,effect in optimizing teaching content and methods,and construct stereo platform including information system,online course,laboratory equipments,research activities and teacher training program.The results showed a significant improvement of the students' knowledge mastering and utilizing capability.And the teaching situation was well re-created.Furthermore,the teaching team was much more powerful than ever.It suggests that ‘three-highlight and stereo' teaching style is a successful explore and practice of teaching reform of laboratory diagnostics on adapting for internationalization of education tendency.
ABSTRACT
Objective To detect the isoniazid (INH) and rifampin (RIF) resistance of Mycohaeterium tuberculosis isolates in the single tube with multiplex-polymerase chain reaction-single strand conformation polymorphism(muhi-PCR-SSCP) system. Methods According to the sequences of inhA, katG and rpoB genes of the Mycohacterium tuberculosis, three pairs of oligonucleotide primes were designed to examine the INH and RIF resistance with the multi-PCR-SSCP. The validity of the newly developed method was evaluated with 116 clinical isolates of Mycohacterium tuberculosis( 70 isolates that were INH-resistant and 66 isolates that were RIF-resistant). Results The validity of the method was assessed with multiplex PCR-SSCP with the bacteria culture with susceptibility test as golden standard. The three genes, katG, inhA and rpoB, in the 116 clinical isolates and H37Rv strain were amplified successfully in single PCR reactions,except 4 isolates with katG deletion mutants. Compared with strain H37Rv, forty-six isolates had katG gene mutations, thirteen had inhA mutations and fifty-eight had rpoB mutations. Thirty-eight isolates had simultaneous katG and rpoB mutations and 4 isolates had both inhA and rpoB mutations. Four isolates had inhA and katG mutations and 2 isolates had mutations in all three genes simultaneously. The sensitivity of the newly developed multiplex-PCR-SSCP assay was 80% and 82% for INH and RIF, respectively. The specificity of the assay was 100% and 92% for INH and RIF, respectively. Conclusion Muhiplex-PCRSSCP provides a rapid, specific and cost-effective method of detecting multidrug-resistant TB. It laid a solid foundation for the further study of drug resistant gene.
ABSTRACT
Objective To construct fusion gene and prokaryofie expression plasmid encoding the Rv1884c and Rv0867c genes in Mycobacterium tuberculosis (M.tb).The fusion protein wsg expressed efficienfly in E.coli cells.Methods The Rv1884c and Rv0867c genes were amplified by polymerase chain reactions (PCR) with specific primers from genomic DNA of M.tb H37Rv strain,and cloned into pGEX-4Tland pUC19 vectors.Rv1884c and Rv0867c were subcloned into the expression vector pGEX-4T-1 and pPRO-EXHT followed by DNA sequencing.The plasmids were transformed into E.coli DH5α and induced to produce GST-fused Rv1884c and His-fused Rv0867c fusion protein.The protein molecular weight and expression format was analyzed by SDS-PAGE.Results The recombinant expressive vectors pGEX-4T-1Rv1884c and pPRO-EXHT-Rv0867c were constructed.The DH5α strains of E.coli with recombinant plasmid showed high level of Rv1884c and Rv0867c gene expressions after IPTG induction.The SDS-PAGE showed that the plasmids expressed Rv1884c and Rv0867c fusion proteins with molecule weight of 45 000 and 80 000.The recombinant protein accounted for 18.3%and 23.7%of total bacteria protein.The expressed proteins could be purified via GSTrao FF and Ni2+-NTA system kits in denatured condition.Conclusions Mycobacterium tuberculosis Rv1884e and Rv0867c genes have been cloned and expressed Successfully in E.coli DH5α.The results lay a basis for further study of fast culfivation in Mycobacterium tuberculosis and investigation of their activities and functions.
ABSTRACT
Objective:To construct a short hairpin RNA(shRNA) eukaryotic expression vector of the survivin gene,and investigate its inhibitory effect on the survivin expression in human cervical cancer cells.Methods:We designed and synthesized 2 pairs of survivin-specific small interfering RNA primers(s1 and s2),cloned them into the eukaryotic expression vector pSilencer 2.1-U6 neo by DNA recombined techniques after annealing connection reaction,and transfected them respectively into human cervical cancer cell line HeLa using LipofectAMINE2000 after identificationby restrict endonuclease digestion and DNA sequencing.Then we selected the positive clones by G418 and detected the expression of survivin mRNA by semi-quantitative RT-PCR and that of the survivin protein by Western blot.Results:The survivin shRNA eukaryotic expression vectors pSilencer2.1-s1 and pSilencer2.1-s2 were successfully constructed.Positive clones were obtained by screening with G418 for 24 days,the survivin expression in HeLa cells decreased in different degrees after transfected with pSilencer2.1-s1 and pSilencer2.1-s2,and the latter showed a better interference efficiency.Conclusion:The shRNA eukaryotic expression vector of the survivin gene we constructed,with its improved interfering efficiency,has paved the way for further research on its role in regulating the biological behavior of cervical cancer cells.
ABSTRACT
Objective: To clone human carboxypeptidase A1 and its active center gene as well as construct recombinant vector for expression before analysis their activities. Methods: CPA1 and CPAlactive center gene were amplified by RT-PCR from pancreas tissue, and then sequencing was carried out. The correct target genes were cloned into prokaryotic vector pGEX-4T-1 and transformed into E. coli BL21 before sequence analysis. After induced by IPTG, gene products were analyzed by SDS-PAGE. Target expressed proteins were denaturated, renaturated, purified and evaluated through MTT and agar colony form test. Results: Human carboxypeptidase Al and its active center gene were cloned successfully. New expected protein band of Mr 66,000 and 46,000 appeared on SDS-PAGE after inducement. Both of the expressed proteins have catalytic activity in vitro, but the activity of the latter is inferior when applied to tumor cells. Conclusions: Human carboxypeptidase A1 and its active center gene were cloned successfully. Their prokaryotic expression products were obtained too. The expressed proteins have catalytic activity in. vitro. A new prosperous beginning of further improvement for CPA1 therapy system has been established based on CPA1 and its active center gene in terms of ADEPT against prostate cancer to clinical application.