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Recombinase polymerase amplification (RPA) is a novel technology for nucleic acid isothermal amplification. It can achieve the rapid amplification and detection of a target gene under 37-42 ℃. This amplification method is highly sensitive, more specific and less instrument-dependent than other existing methods, and it can also integrate multiple detection modes. Therefore, it is especially suitable for applying in low-resource settings and conducting point-of-care tests. Starting from the reaction principles and the experimental design of RPA, this article pointed out some key points when using RPA in a clinical setting. The current development and related problems of RPA were concluded and the various future uses of this method were also prospected.
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Objective To investigate the autophagy level of peripheral blood neutrophils in patients with tuberculosis infection.Methods According to the standard for tuberculosis diagnosis, the subjects were divided into normal control group (16 cases) and active tuberculosis group (24 cases), and peripheral blood was collected.The autophagy of neutrophils and mononuclear cells of the two groups were detected by flow cytometry:The total RNA of neutrophils was extracted by Trizol and the relative quantification of Beclin1 expression was calculated by using 2-△△Ct in real-time quantitative PCR.Results There was no significant difference in age between the active tuberculosis and normal control group (P=0.165 5, P＞0.05);the proportion of male in active tuberculosis group (79.2％) was significantly higher than that of normal control group (37.5％), and the difference was statistically significant (P=0.031 5, P＜0.05).The level of neutrophil autophagy in the active tuberculosis group was significantly lower than that of the normal control group (P=0.013 8, P＜0.05), and there was no difference of mononuclear cells between the two groups (P=0.784 2, P＞0.05);active tuberculosis group the mRNA expression of Beclin1△ Ct=-1.254±0.40 was lower than that of the normal control group△Ct=-0.10±0.48, the difference was statistically significant (P＜0.001).Conclusion The incidence of active tuberculosis in male is higher than that of female;the autophagy level of neutrophils in peripheral blood of patients with tuberculosis infection decreased, and the decline of autophagy-related gene Beclin1 mRNA expression may be related to the occurrence and development of tuberculosis.
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Objective To investigate the distribution and changes of microorganisms on the skin of submariners under a chronically closed environment , and provide reference for targeted medical support .Methods One hundred and twenty-two samples were collected using swab.After culture and isolation, the microbes were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry ( MALDI-TOF-MS) .Results A total of 52 types of 229 bacteria and 2 types of fungi were isolated . Major opportunistic pathogens included Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter cloacae, while major dwelling bacteria included Micrococcus luteus, Oslo Mora bacteria, Acinetobacter, Staphylococcus epidermidis, and Serratia marcescens.Compared with the early period of the task, major opportunistic pathogens and dwelling bacteria were significantly increased in the middle and late period of the task .Conclusion The skin microbes of the submariners are investigated , targeted drugs need to be prepared for daily medical support and war trauma .
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Objective To study the performance verification of lactate dehydrogenase(LDH) in the Johnson Vitros 5 .1 FS bio‐chemical analyzer .Methods According to CLSI instrumentation evaluation standard and referring to the validation scheme provided by the Johnson company ,the precision ,accuracy ,linear range of LDH ,maximum dilution degree ,biological reference range were verified .Results The LDH intra‐batch and inter‐batch precision experiments were≤3 .30% ;the accuracy experiment≤4 .00% ;the determination coefficient of the linear experiment was 0 .997 2 ;the LDH maximum dilution degree was 8 times with saline solution dilution;the biological reference range experiment verified that the reference range 313-618 U/L provided by the VITROS Meth‐odology Manual could be quote .Conclusion The performance verification of LDH detected by the Johnson Vitros 5 .1 FS biochemi‐cal analyzer basically conforms to the requirements of the quality objectives and manufacturer′s instructions ,and meets the needs of clinical test .
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Clinic genetic testing have make rapid progress in recent years , and have been widely applicated in genetic disease diagnosis , infection disease diagnosis and personalized treatment .The aim of this article is to pay attention to the ethical issues raised by genetic testing .The important ethical problems are proposed accompany with prenatal , adolescent, and adult genetic testing respectively .We should adopt responsible best ethical practices in the provision of genetic testing , promote the widely application of gene detection in clinic orderly.
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Objective To study the asymmetric dimethylarginine(ADMA) concentrations in plasma of acute coronary syndrome (ACS) and its various subgroups ,and analyze the relationship between ADMA and traditional risk factors of ACS .Methods 101 ACS patients were enrolled in the study ,which were divided into several subgroups according to clinical diagnosis ,ECG and coro‐nary angiography .ACS group were divided into unstable angina(UA) group(n=70) and acute myocardial infarction(AMI) group (n=31) ,then AMI group was divided into non‐ST segment elevation myocardial infarction(NSTEMI) group(n=19) and ST seg‐ment elevation myocardial infarction (STEMI) group(n=12) .The control group(n= 40) were recruited in the study during the same period ,who showed normal coronary arteries in coronary angiography .All the patients received coronary attery angiography . The plasma ADMA concentration was measured by using enzyme‐linked immunosorbent assay (ELISA) ,and serum contents of lip‐id(TC ,TG ,HDL‐C ,LDL‐C) ,C‐reactive protein(CRP) ,troponin were detected by automatic biochemic analysis apparatus .Results Compared with the control group ,plasma ADMA concentration of ACS group increased ,the difference was statistically significant (P<0 .05) .The concentrations of ADMA in AMI group and UA group were higher than those in control group ,and the difference was statistically significant(P< 0 .05) .The concentrations of ADMA in AMI group were significantly higher than those in UA group(P<0 .05) .Plasma ADMA negatively correlated with HDL‐C concentrations(r= -0 .222 ,P<0 .05) ,positively correlated with LDL‐C concentration(r=0 .357 ,P<0 .05) ,and positively correlated with CRP(r=0 .619 ,P<0 .05) .Conclusion The occur‐rence of ACS and some of its related risk factors are correlated with plasma ADMA concentration .
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Objective To investigate the relationship of serum ferritin(SF) and acute coronary syndrome(ACS) .Methods ACS group(n= 110) was divided into unstable angina(UA) subgroup(n= 46) ,non-ST-segment elevation myocardial infarction (NSTEMI) subgroup(n=30) and ST-segment elevation myocardial infarction(STEMI) subgroup(n=34) according to clinical da-ta ,coronary angiography and electrocardiogram examination .The normal coronary patients provided as control group(n=42) .The levels of serum ferritin ,hs-CRP and serum lipids were determined and the differences of SF among the groups were analyzed .The correlation of hs-CRP ,TG ,LDL-C ,HDL-C ,TC and SF were also analyzed .Results Compared with control group ,the level of SF in UA ,NSTEMI ,STEMI were significantly increased(P<0 .05) .Comparison in the levels of SF in NSTEMI ,STEMI group and UA group had significant difference(P<0 .05) .Correlation analysis indicated that the level of SF were positively correlated with hs-CRP ,TG and negatively associated with HDL-C .Conclusion The level of SF significantly increased in patients with ACS and has a positive relationship with hs-CRP and the disorder of lipid metablism .SF helps in assessing the clinical classification and risk strati-fication in patients with ACS .
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Objective To Investigate the expression of IL-21 and Blimp1 mRNA in Rheumatoid arthritis ( RA) patients and the influence on the expression of Blimp 1 in peripheral blood mononuclear cells (PBMCs) of RA patients after IL-21 stimulated; To further explore the mechanism of IL-21 and blimp1 in the pathogenesis of RA.Methods Case control study.The samples of peripheral venous blood from 50 RA patients of department of rheumatology of The First Affiliated Hospital of Zhengzhou University and 50 healthy people were collected respectively , then the plasma and PBMCs was separated.IL-21 in plasma was measured by ELISA; the correlation between patients clinical index DAS 28, anti-CCP antibody and IL-21 was analyzed.Blimp1 mRNA of patients′PBMCs was detected by qPCR; PMBCs were isolated from RA patient and then cultured in vitro.Blimp1 mRNA level was measured by qPCR and the ratio of CD 20 positive B cell and the ratio of CD138 positive cells in all groups were detected by flow cytometry after 72 h stimulated by IL-21 and CD40L.Results IL-21 content in RA patient blood plasma (130.51 ±11.35)ng/L was significantly higher than that in healthy control (25.46 ±6.05)ng/L, t=5.39,P<0.05.Besides, IL-21 level also had a close relativity with patients DAS28(r=0.658) and anti-CCP antibody (r=0.674, P=0.039 and 0.035).In addition, the expression level of Blimp1 mRNA in RA patient PMBCs (1.321 ± 0.11)was higher than that in healthy control group (1.000 ±0.000), Z=-2.48, P<0.05.While after IL-21 and/or CD40L stimulation, Blimp1 mRNA of IL-21 group and CD40L+IL-21 group(1.084 ±0.029, 1.157 ±0.028)were higher than those of control (1.000 ±0.000)(P=0.002,P=0.001), moreover the expressive level of Blimp1mRNA of CD40L+IL-21 group was higher than that of control group (t=4.862, P=0.02).Compared to negative control group , the ratio of CD20 positive B cells [2.42 ±0.35, 2.63 ± 0.33, 6.35(4.85,6.57),F=278.363,P<0.001] and the ratio of CD138 positive cells(0.474 ±0.110, 0.668 ±0.120, 0.955 ±0.170,F=49.01, P<0.001) in CD40L group, IL-21 group and CD40L+IL-21 group were much higher and the differences among CD 40L+IL-21 group with CD40L group and IL-21 group were statistically significant.Conclusion IL-21 could promote the level of Blimp 1 mRNA in peripheral blood mononuclear cells in RA patient; IL-21 and CD40L could co-promote B cell maturation though regulating Blimp1 mRNA expression and eventually participate in RA pathogenesis.
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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.
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Objective To analyze Blimp1 expression difference in the latent and active of tuberculosis patients and the healthy , and assess possibility of as the new tuberculosis diagnostics molecules .Methods 60 patients with active TB (active tuberculosis group) ,50 participants with latent(latent tuberculosis group) ,and 50 healthy people(control group) were enrolled separately .Using fluorescence quantitative polymerase chain reaction were used to determin Blimp 1 mRNA expression in the peripheral blood mono-nuclear cells .Results Blimp1 mRNA expression level of the active tuberculosis group was 15 .35 times than the control group ,and 2 .21 times than the latent tuberculosis group ,the difference was statistically significant (P<0 .05) .Conclusion Blimp1 gene proba-bly plays a role in the immune response to tuberculosis .it provides new ideas for the laboratory diagnosis of tuberculosis and new clues for further exploring the pathogenesis mechanism of tuberculosis .
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Objective To investigate the type of gene mutation and its distribution in patients with thalassemia in Chongqing ,in order to guide prenatal and postnatal care .Methods PCR and membrane hybridization technology for α-thalassemia andβ-thalasse-mia gene detection .Results From September 2012 to September 2013 ,349 thalassemia patients were detected ,including 125 α-thalassemia patients and 211β-thalassemia patients .Based on genotypes ,gene deletions were the most common type of gene muta-tion of α-thalassemia patients .Southeast Asia deletion - -SEA/αα(accounted for 73 .60 % ) and the right deletion -3 .7/αα(ac-counted for 19 .20% ) were the major types of gene deletion .Forβ-thalassemia patinets ,the hot spots of mutation were CD17(A→T) ,CD41-42(-TCTT) and IVS-Ⅱ-654(C→ T) ,which accounted for 29 .38% ,28 .91% and 27 .49% respectively .α-thalassemia combined with β-thalassemia mutations were detected in 13 patients .Conclusion Studing the gene mutation types of thalassemia and its distribution provides valuable information for genetic counseling and clinical therapy in Chongqing .
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Objective To investigate the significance of the detection of cytomegalovirus (CMV) infection after kidney transplantation. Methods The serum CMV antibody(anti CMV) and CMV DAN of 72 recipients before and after transplantation were detected respectively by enzyme linked immunosorbent assay(ELISA) and polymerase chain reaction(PCR). Results The levels of serum CMV antibody and CMV DNA of 72 recipients after transplantation were significantly higher than those of pre operation( P
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Objective: To construct a lentiviral expression vector that inhibits enhanced green fluorescent proteins(EGFP).Methods: The EGFP shRNA was cloned into the pLL3.7 lentivirus vector digested with XbaⅠ/XhoⅠ.J558L and Hela cells were infected by lentiviruses,and the EGFP expression was observed with the fluorescence microscope.Results: The EGFP RNAi sequence was correctly cloned into the lentivirus vector with the U6 promoter,and the EGFP expressions of Hela and J558L cells were suppressed after infected by lentivirus containing EGFP shRNA. Conclusion: The lentivirus vector effectively inhibited EGFP expression,which could be used to mediate further researches on gene function.
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Objective To probe into the relationship between the viral encephalitis and herpes viral infection, to understand the primary patheogeny of herpes viral encephalitis.Methods HSVⅠ-DNA,HSVⅡ-DNA,VZV-DNA,HCMV-DNA,EBV-DNA in CSF from viral encephalitis patients were detected by PCR.Results 82(15 6%) cases of 526 patiens with viral encephalitis were diagnosed as herpes simplex viral encephalitis, of which the age of high morbidity was at 0～10 years old children,9(1 7%) cases as cytomegaloviral encephalitis,7(1 7%) cases as varicella-zoster viral encephalitis and 4(0 7%) cases as EB viral encephalitis.Conclusions Herpes simplex viruses are the primary pathogeny in herpes viral encephalitis, PCR technique is valuable for the diagnosis of herpes viral encephalitis.