Objective:Using PCR to amplify filamentous fungi DNA fragments. Methods:Pour some liquid nitrogen to the hypha and grind them to powder. Then add some TE buffer and boil them for 15-20 minutes. Take a little supernatant as PCR template. After PCR do agarose gel electrophoresis. Collect the gel containing the objective fragment and add some TE. Take a little supernatant as PCR template (again.) After PCR, do agorose gel electrophoresis again. Results:We obtained a great deal of objective DNA. Conclusion:Amplifying filamentous fungi DNA fragment by this way, we can save time and money. We can also improve our work efficiently.

Objective To establish a multiplex polymerase chain reaction (PCR) assay for simultaneous identification of Shigella spp., Salmonella spp. and Vibrio cholerae. Methods Based on the gene sequences of invasion plasmid antigen H (ipaH) in Shigella spp., invasion plasmid antigen B(ipaB)in Salmonella spp. and enterotoxin extracellular secretion protein (EPSM) in V. cholerae, three pairs of primer were designed. Genomic DNA was extracted by the boiling method and multiplex PCR was performed with premix Taq in an ABI 2720 thermal cycle. The PCR-amplified products were then analyzed by using agarose gel electrophoresis. Results Under the optimized conditions, the assay yielded a 606-bp product from Shigella spp., a 314-bp product from Salmonella spp., and a 482-bp product from V. cholerae, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons in different size were observed. Conclusions A rapid, specific and sensitive multiplex PCR system for simultaneous detection of Shigella spp., Salmonella spp. and V. cholerae has been established. The results suggest that the simultaneous amplification of several genes by multiplex PCR may provide an efficient and rapid diagnostic method for severe diarrhea.

This paper introduces such information of a urinary sediment diagnostic system and its data management system saving data directly from urine analyzer and microscope as its hardware organization and software design.With the performances of easy tooperate and low cost,the system can be applied toclinical urine diagnosis and togenerating standardized reports,whose data management system has high security and accuracy for saving data,strong anti-interference capability,reliable performance,noerror in proceeding and Chinese interface.

HMGB-1, an ubiquitously expressed 25-KD nucleoprotein among mammals, belongs to the HMG family. Recent studies have identified that HMGB-1 is secreted by activated monocytes/macrophages via a non-classical ,vesicle-mediated secretory pathway. Extracellular HMGB-1 is an important proinflammatory cytokine. It participates in the pathogenesis of many diseases such as rheumatoid arthritis, sepsis , acute lung injury, and even leads animals death. Further studies of the mechanism and function of extracellular HMGB-1 may provide a novel strategy for the diagnosis and treatment of these diseases.

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.

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.

Objective To investigate the correlation between the results of HBsAg positive results among different detection systems,and provide reference for the analysis of clinical test results and the publication of the report.Methods The HBsAg positive serum specimens with the quantitation result lower than 80 COI were detected by Roche Cobas e602 electrochemical luminescence analyzer.All the specimens were also detected by Roche Modular e170 electrochemical luminescence analyzer.The differences of detecting results were compared and performed the linear correlation analysis.Results The experimental results of two detection systems are R2 =0.933.The positive coincidences of Group A,B,C,D and E were 60.60％,92.72％,96.66％,96.66％ and 100％,respectively.The positive coincidence of the males was 87.66％,while the positive coincidence of the females was 70.96 ％.The positive coincidence of the males was significantly higher than the females (P＜0.05).Conclusion The HBsAg detecting results of Roche Cobas e602 and Roche Modular e170 electrochemical luminescence analyzer had high correlation.The results higher than 10 COI had 100％ positive coincidence rate,however the results between 1 and 10 COI were not.The result between 1 and 10 COI may lead to controversial results,suggestions for further checks.

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.

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.

Objective To investigate the correlation of HCV-RNA with detection indexes HCV-Ab and HCV-cAg in its clini-cal application effect among patients with hepatitis C.Methods HCV-cAg and HCV-Ab in 140 cases of HCV-RNA were detected by enzyme linked immunosorbent assay in cases of PCR,which were detected by real-time fluorescence quantitative PCR.Results 127 cases in 140 cases of HCV-RNA positive serum were HCV-cAg positive,in line with the rate of 90.71%,and the cases of 110 HCV-Ab positive,in line with the rate of 78.57%.The positive detection rate of HCV-cAg with different HCV-RNA concentration was increased with the increase of HCV virus content,and the serum of different HCV-RNA concentration had no significant changes in HCV-Ab detection results.Conclusion The detection results of HCV-cAg had a high coincidence rate with HCV-RNA.Therefore detection of HCV-cAg can be as a complementary detec-tion of HCV-Ab,as the window period of HCV infection and infection in immunocompromised persons screening provides a simple,inexpensive method.At the same time it provides rapid screening for HCV infection provide diagnostic basis for those basic medical units who do not have the conditions for detection of HCV-RNA.