A preliminary study on the potential value of QuantiFERON-TB Gold Plus(QFT-Plus) in identification of latent tuberculosis infection and active tuberculosis / 中华检验医学杂志

Objective:To evaluate the potential differential diagnostic value of QuantiFERON-TB Gold Plus(QFT-Plus) in patients with active tuberculosis (ATB) and latent tuberculosis infection (LTBI) people.Methods:Case-control study. A total of 108 healthcare workers and 30 ATB patients in Xi′an Chest Hospital were tested by QFT-Plus from April to November 2019, and the demographic characteristics were analyzed.Then, flow cytometry was used to analyze the relations between the QFT-Plus TB2-TB1 and the distribution of peripheral blood T lymphocyte subsets in ATB patients with positive culture results.Finally, with 34 QFT-Plus positive volunteers as LTBI group and 30 bacteriologically confirmed ATB patients as ATB group, the QFT-Plus new lyadded antigen and its potential differential diagnostic value between LTBI and ATB groups was evaluated by using the receiver operating curve (ROC).Results:In patients with ATB,QFT-plus TB2-TB1 was positively correlated with the proportion of CD8+T cells in peripheral blood T lymphocytes( r=0.586, P=0.004), negatively correlated with the proportion of CD4+ T cells( r=-0.511, P=0.015) and the ratio of CD4/CD8 ( r=-0.520, P=0.013).The peripheral blood TB2-TB1 in the ATB patients was significantly higher than that in the LTBI group[0.47(0.12,1.17) IU/ml versus 0.01(-0.08,0.22) IU/ml, U=233.5, P<0.001]. QFT-Plus TB2-TB1 can effectively distinguish ATB from LTBI, with an area under the ROC curve of 0.771 (95 %CI=0.653-0.889, P<0.001). Conclusion:QFT-Plus specific CD8 response (TB2-TB1) has the potential value to identify ATB from LTBI people.

Differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell / 中华检验医学杂志

Objective Studying the differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell,in order to further elucidate the mechanism of androgen-independent prostate cancer and find new targets for its treatment.Methods Prostate cancer androgen-dependent LNCaP cell and prostate cancer androgen-independent LNCaP-AI + F cell (induced by androgen and flutamide) were selected as the study subjects.Illumina HiSeqTM 2500 was used to perform high-throughput sequencing between the two groups.The differentially expressed exosomal miRNAs was verified by quantitative real-time PCR(qRT-PCR).The difference was statistically significant by t-test.Results Through the analysis of high-throughput sequencing results,thirteen molecules were screened increased in extracellular exosomes of the androgen-independent cell,including miR-7-5p,let-7a-5p,miR-375,miR-423-3p,miR-378a-3p,and miR-92a-3p,etc.Among them,miR-7-5p was verified by quantitative real-time PCR to be up-regulated by 19.52-fold (t=9.857,P=0.001).Conclusion Differentially expressed exosomal miRNAs may predict the development of androgen-independent prostate cancer and may further regulate the development of androgen-independent prostate cancer.

Optimization for extraction of urine exosomes and effects of freezing on exosomal RNA content / 临床检验杂志

Objective@#To optimize the existing methods of isolation and purification for exosomes from urine and explore the effects of different storage conditions on the content of exosomal RNA in urine. @*Methods@#The exosomes in human urine samples were extracted by different precipitation method, i.e., precipitation following first concentrating and direct precipitation, respectively, and the separation efficiency and cost of the two methods were compared. ExoQuick-TCTM precipitation kit was used to extract exosomes. Nanoparticle tracking analysis technique (NTA) was used to detect the concentration and particle size distribution of exosome. Dynamic light scattering (DLS) was used to detect the potential of exosome. Transmission electron microscopy (TEM) was used to observe morphology of exosomes. western blot was used to analyze the exosomal marker molecules CD63 and Alix. The extraction method of the precipitation following first concentrating was used to verify the reliability of the optimized method in 10 clinical urine samples . Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of exosomal RNA marker let-7c and PSA mRNA in the urinary exosomes from 20 patients with prostate cancer after repeated freeze-thaw (0 [i.e., fresh], 1 , 3 and 5 times) and 9 patients with prostate cancer frozen at -80 ℃ for different time (0 [i.e., fresh], 1, 2 and 4 weeks), and were statistically analyzed by Wilcoxon rank sum test for differences between the 2 groups. @*Results@#The size distribution of exosomes extracted by the two methods was 30 to 150 nm by NTA, both of which were displayed as single peaks. The results of DLS showed that the potentials of exosome extracted by the two methods were negative values. The size of the exosomes extracted by the two methods was consistent observed under TEM namely the diameter distribution was 30 to 150 nm. western blot analysis confirmed that CD63 and Alix, the exosome labeling molecules, existed in the optimized method. The concentration of exosomes extracted from the 10 urine samples all reached 10 9 to 10 11 particles/mL. The contents of let-7c and PSA mRNA in exosomes decreased significantly after 5 freeze-thaw cycles, and the Z values were -1.79 and -1.73, respectively (P＜0.05). The RNA content of the exosomes remained stable after freezing at -80 ℃ for 1 month. @*Conclusion@#The optimized exosome extraction method could reduce greatly the cost under the premises of ensuring the concentration and quality of exosomes. The isolated exosomes may keep stable RNA content after freezing at -80 ℃ for a short time, but could not be frozen and thawed repeatedly for more than 5 times.

A new perspective of hepatitis markers opened up by exosomes / 中华检验医学杂志

This review, based on the origin and the mechanism of release of exosomes, described the presence of HBV and HCV biomarkers in the exosomes and the mechanisms of secretion, and discussed the viral transmission, vaccination protection, and the influences on treatment and detection.Through basic research data, it preliminarily demonstrated the importance of nucleic acid detection being a gold standard, and pointed out that another new cause of the occult infection was existence of HBV and HCV in the exosomes.(Chin J Lab Med, 2018, 41: 496-498)

Monitoring of blood tacrolimus concentration and lymphocyte subsets in the first uterus transplant recipient in China and its clinical significance / 临床检验杂志

Objective To investigate the changes of peripheral blood tacrolimus concentration and lymphocyte subsets in the uterus transplant recipient,and provide the evidence for monitoring the immune status after uterus transplantation.Methods The peripheral blood tacrolimus concentrations of the uterus transplant recipient during 1 year after transplantation were measured with the microparticle enzyme immunoassay (MEIA).Meanwhile,the whole blood cell counts and lymphocyte subsets were determined by the blood analyzer and flow cytometer,respectively.Results The blood tacrolimus concentrations of the uterus transplant recipient in the first month and second month after transplantation were (13.51 ± 3.92) ng/mL and (15.58 ± 1.19) ng/mL,respectively.The lymphocyte absolute counts were normal before transplantation.At the fifth day after transplantation,the counts of CD3 + T lymphocytes,CD4 + T lymphocytes,CD8 + T lymphocytes and NK cells and the ratio of CD4/CD8 were significantly decreased.One week after transplantation,the counts of CD4 + T lymphocytes were recovered to the normal range and maintained,but its recovery was slower than that of CD8 + T lymphocytes.The ratio of CD4/CD8 ranged from 0.4 to 0.8 during 10 days after transplantation,and increased and maintained between 0.8 and 1.1 after that.The counts of NK cells increased gradually from the 10th day after transplantation,but still did not recover to the level before transplantation even at the 20th day after transplantation.However,the counts and percentages of B lymphocytes did not decrease but increased at the fifth day after transplantation,and recovered to normal gradually from the 10th day after transplantation.There was no significant correlation between the CD3 + T lymphocyte count and blood tacrolimus concentration.Conclusion The dynamic changes of blood lymphocyte subsets and tacrolimus concentration exist in the uterus transplant recipient,which need to be further verified by a large amount of clinical data.

Molecular epidemiological characteristics of Staphylococcus aureus isola-ted in intensive care units / 中国感染控制杂志

Objective To investigate the molecular epidemiological characteristics and homology of Staphylococcus aureus (S. aureus)isolated from patients in intensive care units (ICUs)of a hospital,so as to provide laboratory basis for the effective control of healthcare-associated infection(HAI). Methods 62 S. aureus strains isolated from various specimens from ICU patients with infection in March-August 2013 were collected,7 housekeeping genes were amplified with polymerase chain reaction (PCR),the amplified products were sequenced,ST typing of strains was performed by multilocus sequence typing (MLST ), phylogenetic analysis of ST typing was conducted. Results 62S. aureus strains were amplified specific product of 7 housekeeping genes;there were 10 ST genotypes, in which 2 ST genotypes(STn1and STn2)were first discovered,1 ST genotype(ST675)was first discovered domes-tically. ST239 was the main ST type of S. aureus from ICU patients in this hospital,accounting for 74.20% ,which distributed in 6 ICUs,ST5 distributed in 3 ICUs. 62 strains formed 7 main branches in the phylogenetic tree,55 (88.71% )MRSA strains were detected. Conclusion S. aureus isolated from hospital ICUs has some homology, and the small number of types showed the trend of concentrated distribution.

Exosome-encapsulated miR-375 in urine as a non-invasive biomarker for prostate cancer diagnosis / 中华检验医学杂志

Objective To analyze the expression of urine exosomal miR-375 in prostate tumors and investigate its clinical utility.Methods A total of 45 patients with PCa,24 with benign prostate hyperplasia (BPH) and 24 healthy individuals were enrolled into this study.Exosomes were isolated from the urine of PCa,BPH and healthy individuals and the total RNA was extracted from the exosomes.The exosomal miR-375 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method (2-△△CT).We performed comprehensive biostatistical analyses to explore the clinical value of miR-375 in prostate cancer.Results The urine exosomal miR-375 expression was significantly downregulated in the patients with PCa compared with BPH and the healthy controls (P ＜ 0.01).No statistically significant difference of the urine exosomal miR-375 expression levels between the patients with BPH and healthy individuals was observed (P ＞ 0.05).The urine exosomal miR-375 expression level was also found to be associated with clinical stage and bone metastasis status of the patients with PCa (P ＜0.05),and with the increase of Clinical stage.The expression level of miR-375 decreased.No significant relationship was detected between miR-375 level and the patient's age,gleason score and serum prostate-specific antigen level (P ＞ 0.05).Receiver operator characteristic analyses demonstrated that the urine exosomal miR-375 expression could better differentiate PCa from BPH patients:AUC 0.715 (95％ CI:0.589-0.842) vs PSA AUC 0.632 (95％ CI:0.492-0.771) (P＜0.01).Conclusion The urine exosomal miR-375 could serve as a non-invasive biomarker for the diagnosis of PCa.

Effects of Mycobacterium tuberculosis heparin-binding hemagglutinin(HBHA) on macrophage polar-ization / 中华微生物学和免疫学杂志

Objective To investigate the effects of Mycobacterium tuberculosis heparin-binding he-magglutinin (HBHA) on the polarization of mouse bone marrow-derived macrophages (BMDM) and a mu-rine macrophage cell line(Raw264.7 cells). Methods After HBHA was confirmed to be able to enter into macrophages by immunofluorescence, mouse BMDM and Raw264. 7 cells were treated with LPS (100 ng/ml)+IFN-γ (2.5 ng/ml),IL-4 (20 ng/ml),HBHA (1 μg/ml,3 μg/ml,6 μg/ml,10 μg/ml) and ESAT-6 (5 μg/ml),respectively. IL-6,IL-12 and TNF-α in the supernatants of cell culture were measured by ELISA. RT-PCR was performed to detect the expression of molecular markers of macrophage polarization [inducible nitric oxide synthase (iNOS), TNF-α, Arg-1 and CD206] at mRNA level. Western blot assay was used to detect the expression of iNOS and Arg-1 at protein level. Results Increased secretion of IL-6, IL-12 and TNF-α in the supernatants of cell culture and enhanced expression of iNOS and TNF-α at mRNA level were observed after treating BMDM with Mycobacterium tuberculosis HBHA. Similarly,in HBHA-trea-ted Raw264.7 cells,the secretion of IL-6 and the expression of TNF-α were also up-regulated. Mycobacteri-um tuberculosis HBHA and ESAT-6 had similar effects on murine macrophages. Neither of them could in-crease the expression of Arg-1,a molecular marker of M2 macrophages,in BMDM at protein level,but both enhanced the expression of iNOS,a molecular marker of M1 macrophages,in BMDM and Raw264.7 cells. Conclusion Mycobacterium tuberculosis HBHA can induce the polarization of murine macrophages to M1 phenotype.

Characteristics of antimicrobial resistance and molecular epidemiology of Staphylococcus aureus isolated from patients in intensive care units / 中国感染控制杂志

Objective To study the characteristics of antimicrobial resistance and molecular epidemiology of Staphylococcus aureus (S .aureus)in the intensive care units(ICUs)of a hospital.Methods Clinical isolates of S .aureus collected from ICUs between January and December 2014 were identified and performed antimicrobial susceptibility testing,then typed by staphylococcal protein A (spa)typing and multilocus sequence typing (MLST) methods.Results Of 160 isolates of S .aureus ,120 (75.00%)were methicillin-resistant S .aureus (MRSA). Resistance rates of MRSA to erythromycin,clindamycin,and levofloxacin were all > 80%;methicillin-sensitive S .aureus (MSSA)were sensitive to cefazolin,resistance rates to erythromycin,clindamycin,and levofloxacin were 62.50%,35.00%,and 10.00% respectively.spa typing and MLST results showed that the main types of 120 isolates of MRSA were ST239-t030,ST239-t037,and ST5-t2460,the major epidemic strains were ST239-t030 (n=105,87.50%),and were isolated from 8 ICUs;MSSA had more types,ST59-t437 were detected only from depart-ment of neurology(n =8)and department of digestive diseases(n =2),ST6-t701 ,ST398-t3625,ST398-t1793,and ST121-t2092 were isolated from departments of neurology(n=7),anesthesiology(n=5),neurosurgery(n=4),and cardiac surgery(n=4)respectively.Conclusion Isolation rate of MRSA in ICUs in this hospital is high,ST239-t030 is the main type,which prevailed in hospital;different types of MSSA have epidemic trends in various departments.

The role of microbiome in respiratory disease / 中华检验医学杂志

The development of metagenomics revealed a novel role of microorganism in lots of diseases.Emerging researches at home and abroad illustrated that microbiome changes from nasopharynx, oropharynx and/or lung in quality and/or quantity exist in many respiratory diseases including chronic obstructive pulmonary disease, asthma, cystic fibrosis, pneumonia as well as upper respiratory infection, which play an important role in immune system, metabolism and neuroregulation.These research results may provide us new strategy for the diagnosis, therapy, surveillance and prognosis of respiratory diseases.

Knowledge and factors determining choice of contraception among Singaporean women

<p><b>INTRODUCTION</b>The study aimed to assess the level of awareness and knowledge of contraception among women in Singapore, and identify the factors that influence contraception choice.</p><p><b>METHODS</b>We conducted a cross-sectional survey of 259 female patients, aged 21-49 years, who attended the Obstetrics and Gynaecology Clinic at National University Hospital, Singapore. An original questionnaire on nine contraceptive methods was used. Respondents who had ≥ 2 correct answers for a method (out of four questions) were considered to have good knowledge of the method. Participants were asked to rate factors known to influence contraceptive choice as important or not important.</p><p><b>RESULTS</b>Awareness of the following methods was high: condom (100.0%), oral contraception pill (89.2%), tubal ligation (73.0%) and copper intrauterine device (IUD) (72.2%). The women were least aware of hormonal IUD (24.3%). Women who were parous, had a previous abortion, had completed their family or used contraception previously were more likely to have a higher awareness of contraception. 89.2% of the women had good knowledge of the condom; among those aware of hormonal IUD, only 46.0% had good knowledge of it. Women who had used hormonal IUD and the condom were more likely to have good knowledge of them. Many rated efficacy (90.5%) and a healthcare professional's advice (90.1%) as important in contraceptive choice. Few considered peer influence (21.0%) and cultural practices (16.3%) to be important.</p><p><b>CONCLUSION</b>Women in Singapore have poor awareness and knowledge of contraception, especially long-acting reversible methods. More effective ways are needed to educate women about contraceptive methods.</p>

Isolation of human serum exosome and the clinical value of exosomal miRNA detection / 中华检验医学杂志

Objective To isolate and identify exosomes from human serum , explore the feasibility of detecting exosomal miRNA in human serum.Methods Retrospective study.Serum samples from 10 healthy individuals in January 2013 were randomly selected.Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients ( n=20 ) and healthy controls ( n=20 ) were selected.Exosomes were isolated from these serum samples using ExoQuick , and then identified by using transmission electron microscopy , NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype.The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser.Then quantificational real-time polymerase chain reaction ( qRT-PCR) was carried out to detect miRNAs in different components of human serum ,and nonparametric tests were used for difference analysis.Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm.Moreover, they expressed HSP70 and four transmembrane protein CD 63.Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA.qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome , and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, U =2,P <0.000 1;let-7a, U=13,P=0.003 2) and exosome-depleted supernatant ( miR-21, U=15,P=0.006 5;miR-16, U=2,P<0.000 1;miR-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).miR-141, the molecular marker of prostate cancer ,were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients , 20 BPH patients and 20 healthy control people.The results showed that , in three groups , exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66,P=0.000 3; BPH group, U=83,P=0.001 6;PCa group, U=54,P<0.000 1).In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74,P=0.000 7 and 4.06 fold, U=70,P=0.000 5).Conclusion Exosome can be efficiently isolated from human serum.Compared with the whole serum , isolation of serum exosome may helpful to improve the detection of circulating miRNA.

Quality assurance of clinical biochemistry testing:a mualti-center study based reference interval for clinical chemistry tests in the Chinese population / 中华检验医学杂志

Objective To verify and monitor the performance of accuracy, precision and comparability of 26 clinical biochemical analytes (29 methods) in the six centers involved in multi-centers reference intervals research, and to ensure the reliability of theirmeasurement results.Methods During the period of the systems evaluating, two levels of commercial quality control materials and fresh frozen human serum reference materials were applied to verify the performance of inter-laboratory precision and accuracy of analysis systems. During the period of samples testing, the commercial quality control materials were measured whenever samples were analysed, the fresh frozen serum reference materials were measured once a month.The coefficient of variations (CVs), bias and total errors were calculated to assess the precision, accuracy and comparability.Results Verification of precision and accuracy: ( 1 ) the ranges of CVs of 29 methods in the six laboratory laboratories were 0.4%-6.0%, the CVs of all 29 methods met the criterion . (2) The overall average bias of the analysis systems of 21 analytes (24 methods) ranged from -5.15%( ALT) to 4.46% ( Ur ) .Among 24 methods the overall average bias of TP, Glu-GOD, Ur, Cl, Ca exceeded the acceptable range.The quality assessment during the period of samples testing:(1) The overall average bias ranged from -1.95%(Ca) to 2.92%(Ur), median 1.26%, they all met the requirements of relevant standards.( 2 ) When commercial control materials were tested, the requirements of CVs were fulfilled for most methods in the six laboratories,and the CVs of TP, Alb, Cl, Ca exceeded the acceptable range.The overall average TE of all methods met the quality specification for the C-N controls material.For the C-P control material, only the overall average TE of TP (5.05%) exceeded thearceptable range while the other methods met the requirement in criterion.Conclusions The performance of precision and accuracy of the analysis systems used in the six laboratories passed the verification.During the period of sample testing, the performance of precision and accuracy of the most methods in the 6 laboratories met the requirements of quality specifications, and the overall performance was good.Because of the limitation of current technology the performance of some methods didn't fulfill the requirement of specifications, and need to be improved.

Expression and diagnostic value of HBHA truncated protein / 国际检验医学杂志

Objective To express recombinant HBHAΔC and HBHAΔN protein,and compare the HBHA series protein activity with each other.It will be provide a experimental basis for the research on clinical diagnostic of HBHA.Methods The HBHAΔC and HBHAΔN gene fragments were cloned and expressed by transforming E.coli BL-21.Test the protein heparin binding ability by CL-6B column.And then added protein to the BCG 7H9 culture medium,to observe the induced BCG aggregation.Results nHB-HA,rHBHA and HBHAΔN protein have heparin binding ability.Meanwhile nHBHA,rHBHA and HBHA Δ C protein have in-duced BCG aggregation effect.Conclusion The HBHAΔC and HBHAΔN protein were successfully obtained.It was proved that the HBHA C-terminal could be combined with heparin and the N-terminal involved could induce the aggregation of BCG.This results provide a basis for further study on molecular mechanism of TB infection and clinical application.

"The explore of ""three-highlight and stereo"" teaching style of laboratory diagnostics" / 中华检验医学杂志

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.

Development of diagnostic procedure of TaqMan MGB probe-based real-time PCR for prediction of response to HCV therapy / 中华检验医学杂志

Objective Establishment and development of a novel Single-Nucleotide-Polymorphism TaqMan Real-Time PCR assay for rapid detection of rs12979860 that predicts HCV therapy response.Methods Human genomic DNA were extracted from solid tissues,secretion and plasma before allelic discrimination.With the property of minor groove binding protein (MGB) binding to minor groove of DNA with strong specificity and affinity,primers and MGB probes were particularly designed for differentiation of human genomic frequencies.MGB probe-based real-time PCR was established to increase allelic discrimination using two probes that only differ in one nucleotide of IL28B rs12979860.The specificity was evaluated by fluorescence signal emissions which were selected from two signal channels.And DNA sequencing was used to confirm the genomic polymorphisms.Results TaqMan probe-based SNP real-time PCR increased allelic discrimination using two probes that only differ by one nucleotide of amplicon,which indicated this assay was easily performed regardless of genomic DNA concentration and quality,minimizes sources of error.The sensitivity was as low as 1.5 ng/μl,the amplification efficacy was 97.6％.The genotype frequencies of CC,CT were remarkably different between Caucasian and Mongolian.The dominated genotype of Caucasian was CT,while most Mongolian was carried CC (26/40 vs.40/50,x-2 =18.75,P value ＜ 0.05).However,the genotype between two population showed no relationship with their virological clearance clinically (P value ＞ 0.05).Conclusions This TaqMan MGB assay shows highly specificity,which has potential as a routine diagnostic test for the detection of rs12979860 from various types of samples.This robust assay would be widely used clinically to predict patients' response before anti-HCV treatment in personalized medicine.

The expression of two-component system response regulator in multidrug-resistant Mycobacterium tuberculosis / 中华检验医学杂志

ObjectiveTo screen out the two-component system associated with drug resistance of Mycobacterium tuberculosis by detecting the differential expression of two-component system regulator genes between multidrug resistant Mycobacterium tuberculosis strains and drug sensitive strains. MethodsTotal RNA of MTB was extracted from cultured MTB during the logarithmic phase in the 7H9 brook medium, and then its purity was identified. Reverse transcription was further completed. The expressing levels of TCS response regulators were quantified using SYBR Green I qRT-PCR, which aimed at finding the differential expressions between multidrug resistant strains and sensitive strains. Finally, all of differentially expressed TCS were screened out under the stress of INH, SM and LFA. Results Compared with sensitive strains,multidrug resistant strains of Rv0491, Rv3133c, Rv3143 and Rv3246c were up-regulated 1. 03, 7.11,3.48and 1.37 folds, respectively (t/t' =5. 623, -4. 196, -3. 559 and -3. 016, respectively, P ＜0. 01 ). The expressing level of other regulators had no statistical significance between muhidrug resistant strains and drug sensitive strains. Under the antibiotic pressure, the expression of Rv1027c, Rv3246c and Rv3143 showed significant changes compared with no antibiotic group. ConclusionRv3246c and Rv3143 may be associated with MTB drug resistance and the differentially expressed genes in multi-drug resistant strains may be used as potential drug targets against drug resistant tuberculosis.

Gene mutations analysis in resistant Mycobacterium tuberculosis isolates / 中华检验医学杂志

Objective To investigate the relationship between the phenotypes and the patterns of genetic mutations in the corresponding resistance genes (rpoB, katG, inhA, ahpC, rrs, rpsL, embB and gyrA) in resistant Mycobacterium tuberculosis (MTB) isolates. Methods Rifampicin-resistant gene (rpoB), isoniazid-resistant genes (katG, inhA, ahpC), streptomycin-resistant genes (rrs, rpsL), ethambutol-resistant gene (embB) and quinolinone-resistant gene (gyrA) were amplified by PCR with sequence-specific primers, then mutants screened by single-stranded conformation polymorphism (SSCP) were sequenced. Results rpoB mutation with predominant Ser450Trp pattern was 94. 9% (56/59) in 59 rifampicin-resistant isolates;katG mutation rate was 38. 9% (35/90) and the main pattern was Ser315Thr, but only 3 inhA mutants and no ahpC mutation were determined in 90 isoniazid-resistant isolates;gyrA mutation with main Asp94Gly then Ala90Val pattern was 82.4% (28/34) in 34 quinolinone-resistant isolates;the total mutation rate was 77.4% in 31 streptomycin-resistant isolates, of which 15 isolates mutated in rrs with main pattern A514C or A1041G, 10 isolates mutated in rpsL Lys88Arg;and embB mutation with main Met306Val accounted for 19.4% (6/31) in 31 ethambutol-resistant isolates. Conclusions The results showed that resistance of resistant MTB may be complicated, and DNA sequencing-based mutation analysis could efficiently detect the molecular makers such as rpoB, katG, gyrA, rrs, rpsL and embB in resistant MTB isolates. Meanwhile, it is notable that the rpoB mutation pattern in our isolates is different from previous report, further effort are needed to confirm the characteristics. The spectrum of potential resistance-related mutations in MTB clinical isolates may lay substantial foundation for the rapid molecular diagnosis and rational use of drug to MTB patients.

Purification and activity determination of native and recombinant HBHA protein / 中华检验医学杂志

Objective To purify native and recombinant heparin-binding hemagglutinin(HBHA)protein,and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus CalmetteGuerin(BCG)induced by HBHA.Methods After growing BCG to the stationary phase in the 7H9 liquid medium,the native HBHA protein(nHBHA)was obtained by CL-6B column chromatography.At the same time,the HBHA gene fragment was cloned and expressed by transforming Escherichia coli BL-21.Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit.Different comcentration of the HBHA protein was added to the BCG liquid medium,and the aggregation of the BCG was observed.Then,add the HBHA protein that incubated with anti-HBHA antibodies to the BCG culture medium and observe the aggregation of BCG.Results The purity of native HBHA was 99% and the concentration was 1.016 mg/ml.The expressed product contained 36% of total somtic protein.After purified,the purity of the recombinant HBHA protein was 97.1% and the concentration was 10.98 mg/ml.Both the rHBHA and nHBHA could induce the aggregation of BCG.When then concentration of nHBHA is 0.2μg/ml,BCG could be induced to aggregate,while the rHBHA concentration is 2μg/ml could induce the aggregation.Both aggregations could be suppressed by the polyclonal antibody against rHBHA.Conclusions The native and recombinant HBHA are successfully obtained.It is proved that the rHBHA could induce the aggregation of BCG similar as nHBHA,and polyclonal antibody against rHBHA could also suppress the activity of nHBHA.It suggested that rHBHA could be further used in clinical diagnosis and vaccination.

Effects of Micrococcus luteus Rpf and Rpf domain protein on growth of Mycobacterium tuberculosis / 中华微生物学和免疫学杂志

Objective To purify Micrococcus luteus Rpf and Rpf domain fusion protein, and to in-vestigate its effects on growth of Mycobacterium tuberculosis. Methods The recombinant plasmids pPro-EXHT-Rpf and pPro-EXHT-Rpf domain were expressed in E. Coli DHSa and then purified under denaturing condition via Ni-NTA purification system and confirmed by Western blot. The biochemical property of the M. Luteus Rpf and Rpf domain was analyzed by stimulating the resuscitation of M. Tuberculosis H37Ra which were in non-culturable' condition. Results The Rpf and Rpf domain products achieved 95% and 93% pure respectively, and the molecular weight was 30 x 103 and 12 x 103, the yield of purification was about 471 mg/L and 337 mg/L of the culture. The M. Luteus Rpf and Rpf domain from the E. Coli showed activity of stimulating the resuscitation of M. Luteus and M. Tuberculosis H37Ra in non-cuhurable' condition which could be inhibited by monoclonal antibodies of M. Luteus Rpf domain remarkably. Conclusion It was dem-onstrated that the purification of Rpf and Rpf domain have high biological activity for further functional, pharmacological and clinical investigations, and M. Luteus Rpf domain protein is fully active as M. Lateus full-length Rpf.