Objective To establish a molecular inversion probe ( MIP) method for detection of single base drug-resistance mutation in Hepatitis B virus ( HBV) gene.Methods The HBV wild type and YVDD mutant strain were isolated by Daping Hospital of the Third Military Medical University.The MIP was designed and applied to detect the HBV drug-resistance YVDD mutation in one case of wild type and one case of YVDD mutant HBV strain isolated previously.The results of MIP method were compared with that of sequencing to evaluate the detection accuracy.Results Thermal cycling single-base extension and connection reaction performed by Taq DNA Ligase and Ampligase DNA Ligase could ensure the specificity of the detection.The optimum probe concentration of MIP was 1 nmol/L.Through detection of the target gene with different DNA concentrations , the detection sensitivity of MIP was determined as 1 nmol/L.The results of MIP were consistent with that of sequencing method in detection of the clinical samples.Conclusion MIP is successfully used to detect single-base drug-resistance mutation in HBV gene.

Objective To investigate the gestation-specific reference intervals (GRIs) and dynamic changes of thyroid function at different gestational ages in Chongqing .Methods Combining self-sequential longtitudinal with cross-sectional study, the serum samples from 640 pregnant women with different gestational age were collected from June 2014 to September 2015 in the Third Military Medical University. The free triiodothyronine (FT3), free thyroxine (FT4), thyroglobulin (TG), thyroid-stimulating hormone (TSH), thyroid peroxidase antibody (TPOAb), and antithyroglobulin antibody (TGAb) were detected by the direct chemiluminescence method.According to China Guideline for the diagnosis and treatment of thyroid disease in pregnancy and postpartum in 2012, the reference interval of the thyroid function was calculated.The data were analyzed by Chi square test .Results Established GRIs of thyroid function during pregnancy in Chongqing:The GRIs was 3.68-5.59 pmol/L for FT3, 9.34-17.02 pmol /L for FT4, 0.18-5.26 mIU/L for TSH in 6-9+6 weeks of pregnancy; the GRIs was 3.69-6.03 pmol /L for FT3, 8.42-15.75 pmol/L for FT4、0.09-4.85 mIU/L for TSH in 10-13+6 weeks of pregnancy; the GRIs was 3.24-5.46 pmol /L for FT3, 6.50-14.24 pmol/L for FT4, 0.11-5.13 mIU/L for TSH in 14-27+6 weeks of pregnancy;the GRIs was 3.06-5.05 pmol /L for FT3, 6.12-11.69 pmol/L for FT4, 0.75-3.67 mIU/L for TSH in 30-34 weeks of pregnancy; the GRIs was 2.96-5.00 pmol/L for FT3, 6.26-11.36 pmol /L for FT4, 0.84-5.54 mIU/L for TSH in 36-40 weeks of pregnancy.Screening by GRIs, the prevalence of thyroid dysfunction was 8.75% (46), however, the prevalence was 37.07% (195) in according with the guidelines,χ2 =120.5,P =0.000.The overdiagnosis rate was 28.32%(149 /526).Using the guidelines of thyroid disease and our GRIs, the thyroid disease was found 116 (22.05%) and 30 (5.70%) in the first screening. Moreover, the thyroid disease was found 79(19.27%) and 10(3.23%) during the repeat screening in the normal population.Conclusions Using the GRIs for thyroid function tests in normal singleton pregnant women could reduce the risk of over diagnosis .The detection rate of repeat screening of TPOAb negative patients was close to the first screening detection rate , and repeated screening could reduce the risk of missed diagnosis for thyroid dysfunction in pregnancy women .(Chin J Lab Med, 2016, 39:511-515 )

To separate and identify chemical signals which induce Thesium chinense haustorium formation, the components of T. chinense roots secretion collected with XAD-4 resin were detected by GC-MS. The effect of DMBQ as exogenous signals to induce haustorium formation in T. chinense was studied. Fifty-three compounds of 9 types had been detected, including hydrocarbons, esters, organic acids, ketones, alcohols, nitrogen containing compounds, phenolic acids, aldehyde and quinine. It is worth noting that the 2, 5-di-tert-butyl-1,4-benzoquinone has the core structure of 1,4-benzoquinone, which may play an important role in the parasitic relationship of Prunella vulgaris and T. chinense: DMBQ worked effectively on inducing haustoria, but induction effects vary widely in different concentrations. DMBQ with the concentration of 1 μmol x L(-1) showed the best effect of the inducing ability with a ratio of 110.52 when treated to induce haustoria.
Gas Chromatography-Mass Spectrometry ; Host-Parasite Interactions ; Magnoliopsida ; chemistry ; physiology ; Plant Roots ; chemistry ; physiology ; Prunella ; chemistry ; physiology

Objective To establish a new method for rapid detection of β-thalassemia by investigating six clinical common mutation types.Methods Fifty cases of clinical wild-type samples and 42 cases ofβ-thalassemia samples were collected, and β-globin gene was amplified by PCR.Uniform ligation probe ( ULP) specific probes were designed for hybridization reaction to increase the reaction specificity and real-time PCR was performed to increase the sensitivity.After that, PCR products were verified by agarose electrophoresis.After examining the specificity and sensitivity, Kappa test between LDR-ULP method and reverse dot blot( RDB) method was conducted.Results Hybridization efficiency was improved 2.53 times by LDR-ULP hybridization.Each mutant type showed a significant amplification curve, whereas the wild-type had no significant curve within 40 cycles.The limit of determination of this method was 5 pg.The results of 92 cases of peripheral blood samples detected by the method of LDR-ULP and RDB were completely consistent.Conclusion In this study, a simple, inexpensive, rapid new method to detect β-thalassemia were established.

Objective To establish a homothermal and fast detecting method on pathogenic bacteria by combining recombinase-aid amplification (RAA) with molecular beacon.Methods The establishment of the methodology.Staphylococcus aureus specific primers were designed from the relative region of the staphylococcal protein A (SPA).Asymmetry amplification was optimized by adjusting the primer concentration ratios.The results of amplification and hybridization were visualized and analyzed by agarose gel electrophoresis and fluorescence detection.The sensitivity was identified by detecting dilute positive plasmids.And the specificity was determined using RAA method by detecting 72 pathogenic bacteria,including Staphylococcus aureus and other Staphylococcus spp.from the Department of Clinical Laboratory of Daping Hospital in December 2016.Besides,the Kappa analysis and the clinical diagnosis efficiency were investigated by analyzing 39 extra strains in the laboratory in December 2016.Results When the concentration ratio of restrictive and non-restrictive primer was 1:20,the yield efficiency of single-stranded DNA (ssDNA) reached the peak.And as for the hybridization efficiency,the asymmetry amplification was higher than symmetry amplification.Twenty copies/μl was proposed as the limits of detection by testing dilute plasmids.And the RAA hybridization method could distinguish Staphylococcus aureus with other Staphylococcus spp.Comparing with traditional detection methods with a Kappa index of 0.860,this method shows a good consistency.By analyzing the 111 bacteria,the sensitivity of the method is 92.5％ (37/40),the specificity is 97.2％ (69/71),the positive predictive value is 94.9％ (37/39),the negative predictive value is 95.8％ (69/72),the positive likelihood radio is 33.04,the negative likelihood radio is 0.077,the Youden index is 0.897 and the Kappa index is 0.902.Conclusion Through the combination of asymmetry recombinase-aid amplification optimization and molecular beacon probe,a new method of detecting bacteria DNA with RAA hybridization technique is established,providing the foundation for its clinical application.

OBJECTIVETo compare the therapeutic effect of posterior lumbar interbody fusion by single and double B-Twin expandable spinal spacer with micro endoscopic discectomy (MED) for lumbar intervertebral disc protrusion accompanying degenerative instability.

METHODSFrom March 2006 to May 2008, 45 patients with lumbar intervertebral disc protrusion accompanying degenerative instability were admitted and managed with posterior lumbar interbody fusion by B-Twin expandable spinal spacer with MED. The patients were randomly assigned to treatment with single B-Twin (Single group, n = 24) or double B-Twin (Double group, n = 21). There were 16 males and 8 females, with an average age of 45.5 years (43 - 60 years) in Single group; 13 males and 8 females, with an average age of 43.7 years (44-61 years) in Double group. All the cases suffered from only one level disc protrusion, L(3-4) 2 cases, L(4-5) 29 cases and L₅-S₁ 14 cases. Clinical outcomes were evaluated with surgical time, blood loss, visual analogue scale (VAS) scores preoperatively, 1, 3, 6 month postoperatively. Oswestry disability questionnaire (ODI) of the preoperative, 1 month postoperative, and latest follow-up and the disk space heights.

RESULTSForty three patients were followed-up for 1 to 3 years after surgery. The mean surgical time of Double group was longer than Single group [(152 ± 32) min vs. (91 ± 15) min, P < 0.01]. The average blood loss in Double group was more than that in Single group [(146 ± 73) ml vs. (95 ± 58) ml, P < 0.01]. The mean time of hospital stay in Single group was similar to that in Double group [(11.0 ± 3.2) d vs. (10.9 ± 3.3) d, P > 0.05]. Both groups could keep the disk space heights till the last follow-up [(7.7 ± 1.8) mm vs. (8.5 ± 1.7) mm]. In the 6 months follow-up post operation, the VAS score decreased from (8.1 ± 1.8) to (2.0 ± 1.0) in Single group, and (8.1 ± 1.9) to (2.1 ± 1.0) in Double group. At the last follow-up, the ODI decreased from (36 ± 7)% to (10 ± 4)% in Single group and (37 ± 6)% to (9 ± 4)% in Double group, but there was no significant difference between the two groups (P > 0.05). All the cases achieved fusion at the last follow-up, 3 patients in Single group and 2 patients in Double group suffered from intractable low back pain. One of the fins broke in one patient without any uncomfortable feeling.

CONCLUSIONSCompared with the management of lumbar intervertebral disc protrusion accompanying degenerative instability by double B-Twin expandable spinal spacer with micro endoscopic discectomy, the single B-twin can get similar clinical outcomes, but shorter surgical time, less blood loss and less medical costs.

Adult ; Endoscopy ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc Displacement ; surgery ; Lumbar Vertebrae ; Male ; Middle Aged ; Prospective Studies ; Spinal Fusion ; methods ; Treatment Outcome

Introduction:The 2010 targets of the China Hepatitis B Prevention Programme were a prevalence of hepatitis B surface antigen (HBsAg) less than 1.0% for children less than five years old and less than 6.0% for the total population. This survey assessed the prevalence of Hepatitis B infection in Lianyungang, Jiangsu province, China in 2009–2010.Methods:Multistage sampling was used with 2372 subjects among 17 selected villages. Blood specimen collection and testing by enzyme-linked immunosorbnet assay (ELISA) were completed using the following markers for hepatitis infection: HBsAg and antibody to HBsAg (anti-HBs); hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe); and hepatitis B core antibody (total anti-HBc). The data were analysed with Epi Info, version 3.3.2.Results:The prevalence of HBsAg was 2.4% (95% Confidence Interval [CI]: 1.8–3.0; Adjusted Prevalence [AP] 2.9%); anti-HBs prevalence was 51.1% (95% CI: 49.1–53.1; AP 49.2%) and total anti-HBc prevalence was 41.7% (95% CI: 39.8–43.7; AP 45.5%). The prevalence of HBsAg and total anti-HBc positivity increased from young to older age groups, yet the prevalence of anti-HBs positivity decreased from young to older age groups (P< 0.001 for all). There was no difference in the prevalences of HBsAg and anti-HBs among females and males (P= 0.108 and 0.089), but females had a higher prevalence than males for total anti-HBc positivity (P< 0.001). Discussion: This survey showed that in 2010 the prevalence of HBsAg among children aged less than five years was lower than the national target of 1.0% and that the prevalence of HBsAg for the total population was lower than the national target of 6.0%.

In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed a novel siRNA expression vector containing multiple tandem siRNA cassettes to investigate the synergistic inhibitory effect of it on human colon cancer cells proliferation. Multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes was constructed and transfected into human colon cancer cell. The effect of multiple siRNA recombinant expression vector was detected by RT-PCR, Western blotting and flow cytometry. It was confirmed by restriction endonuclease and sequence analysis that multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes was constructed successfully. Livin and Survivin genes inhibition ratio of Livin and Survivin siRNA at mRNA levels were 27.90% and 32.24%, at protein levels were 22.28% and 40.86%, the apoptotic ratio was (11.69 +/- 1.37) %, but the synergistic effect was weaker than Livin and Survivin RNA interference, respectively. The multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes has been constructed successfully. It can inhibit the expression of Livin and Survivin genes in human colon cancer cells, but the synergistic effect was weaker than Livin and Survivin RNA interferences, respectively.
Adaptor Proteins, Signal Transducing ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Neoplasm Proteins ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effect of siRNA targeting Livin and Survivin gene simultaneously on the proliferation and apoptosis of human colon cancer cells.

METHODSSiRNA recombinant expression vectors targeting Livin and Survivin gene simultaneously were constructed and transfected into human colon cancer cell line Lovo. The effects of siRNA recombinant expression vector on Lovo cells were detected by RT-PCR, Western blot, MTT reduction assay and flow cytometry.

RESULTSIt was confirmed by restriction endonuclease and sequence analysis that siRNA recombinant expression vector targeting Livin and Survivin gene simultaneously was constructed successfully. The suppressive rates of siRNA targeting Livin and Survivin gene simultaneously on Livin mRNA and protein expression were 27.9% and 22.3% respectively, and those on Survivin mRNA and protein expression were 32.2% and 40.9% respectively. The survival rate of cancer cells was decreased whereas the apoptotic rate was increased, but the coordinate repression was weaker than Livin and Survivin RNA interference alone.

CONCLUSIONSsiRNA targeting Livin and Survivin gene simultaneously can decrease the expression of Livin and Survivin gene, suppress cell proliferation and induce cell apoptosis in human colon cancer. The coordinate repression was weaker than Livin and Survivin RNA interference alone.

Adaptor Proteins, Signal Transducing ; genetics ; Apoptosis ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; pathology ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; RNA, Small Interfering

OBJECTIVETo investigate the effects of small interfering RNA (siRNA) recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil.

METHODSsiRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis.

RESULTSRestriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treating alone (P<0.05).

CONCLUSIONThe siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil.

Antimetabolites, Antineoplastic ; therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; drug therapy ; genetics ; metabolism ; Fluorouracil ; therapeutic use ; Genetic Vectors ; genetics ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism