微流控环介导等温扩增法在脓毒症患者肺炎克雷伯菌检测中的应用价值
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摘要
目的探讨微流控环介导等温扩增法在脓毒症患者肺炎克雷伯菌检测中的应用价值。方法收集2015年7月～2018年5月河北北方学院附属第一医院重症医学科脓毒症患者152例作为研究对象,自然咳痰采集法收集痰液标本99例,经支气管镜抽吸法收集标本53例,分别进行细菌培养及微流控环介导等温核酸检测,根据细菌培养是否分离到肺炎克雷伯菌分为肺炎克雷伯菌组42例和非肺炎克雷伯菌组110例。结果细菌培养检出肺炎克雷伯菌阳性率为27.6%(42/152),微流控环介导等温核酸检测法阳性率为38.8%(59/152),后者阳性率高于前者,差异有高度统计学意义(P<0.01)。肺炎克雷伯菌组经微流控环介导等温核酸扩增法检出阳性菌37例,其中自然咳痰采集法检出21例,支气管镜抽吸法检出16例,非肺炎克雷伯菌组检出阳性菌22例,其中自然咳痰法检出15例,支气管镜抽吸法检出7例,经支气管镜抽吸法和自然咳痰采集法检测的ROC曲线下面积为0.847和0.836,约登指数前者(0.694)高于后者(0.672)。与细菌培养法的一致性评价(Kappa值为0.604>0.600,P<0.05)比较,两种检测方法有较高的一致性。结论微流控环介导等温扩增法在脓毒症患者肺炎克雷伯菌检测中具有快速、灵敏的优势,与传统的细菌培养法有较高的一致性,为临床提供更好的的诊疗依据。
Objective To explore the clinical value of microfluidic loop-mediated isothermal amplification detection in the diagnosis of Klebsiella pneumoniae infection in sepsis patients. Methods From June 2015 to May 2018, 152 sepsis patients who were from the department of ICU of the First Affiliated Hospital of Hebei North University were collected,99 cases of sputum specimens samples were collected by natural cough method, 53 cases were collected by bronchoscopy. All these samples were simultaneously detected by traditional pathogen culture method and microfluidic loop-mediated isothermal amplification method. According to the traditional bacterial culture result, all cases were divided into two group, 42 cases were Klebsiella pneumoniae infection group and 110 cases were non Klebsiella pneumoniae infection group. Results The positive rate of Klebsiella pneumoniae detected by bacterial culture was 27.6%(42/152), and by microfluidic loop-mediated isothermal amplification detection was 38.8%(59/152), which was higher than the former, the difference was statistically significant(P < 0.01). The method of microfluidics loop-mediated isothermal amplification detected 37 positive cases from Klebsiella pneumoniae infection group, including 21 cases natural cough sample and 16 cases bronchoscopy sample, Non Klebsiella pneumoniae infection group was detected by the method of microfluidics loop-mediated isothermal amplification had 22 cases positive samples,including 15 cases natural cough samples and 7 cases bronchoscopy samples. The area under the ROC curve of the bronchoscopy suction method and natural cough collection method was 0.836 and 0.847. The Jordan index of former(0.694) was higher than the latter(0.672). The Kappa value was 0.604 > 0.600, P < 0.05, indicating a high consistency between the two methods. Conclusion Microfluidic loop-mediated isothermal amplification has the advantages of rapid and sensitive detection of Klebsiella pneumoniae in sepsis patients, which is highly consistent with the traditional bacterial culture method and can provide better clinical diagnosis and treatment basis.
Objective To explore the clinical value of microfluidic loop-mediated isothermal amplification detection in the diagnosis of Klebsiella pneumoniae infection in sepsis patients. Methods From June 2015 to May 2018, 152 sepsis patients who were from the department of ICU of the First Affiliated Hospital of Hebei North University were collected,99 cases of sputum specimens samples were collected by natural cough method, 53 cases were collected by bronchoscopy. All these samples were simultaneously detected by traditional pathogen culture method and microfluidic loop-mediated isothermal amplification method. According to the traditional bacterial culture result, all cases were divided into two group, 42 cases were Klebsiella pneumoniae infection group and 110 cases were non Klebsiella pneumoniae infection group. Results The positive rate of Klebsiella pneumoniae detected by bacterial culture was 27.6%(42/152), and by microfluidic loop-mediated isothermal amplification detection was 38.8%(59/152), which was higher than the former, the difference was statistically significant(P < 0.01). The method of microfluidics loop-mediated isothermal amplification detected 37 positive cases from Klebsiella pneumoniae infection group, including 21 cases natural cough sample and 16 cases bronchoscopy sample, Non Klebsiella pneumoniae infection group was detected by the method of microfluidics loop-mediated isothermal amplification had 22 cases positive samples,including 15 cases natural cough samples and 7 cases bronchoscopy samples. The area under the ROC curve of the bronchoscopy suction method and natural cough collection method was 0.836 and 0.847. The Jordan index of former(0.694) was higher than the latter(0.672). The Kappa value was 0.604 > 0.600, P < 0.05, indicating a high consistency between the two methods. Conclusion Microfluidic loop-mediated isothermal amplification has the advantages of rapid and sensitive detection of Klebsiella pneumoniae in sepsis patients, which is highly consistent with the traditional bacterial culture method and can provide better clinical diagnosis and treatment basis.
引文
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[14]李松,何晓光,马可泽.环介导恒温扩增芯片法在机械通气新生儿下呼吸道病原菌检测中应用[J].广东医科大学学报,2018,36(2):169-172.
[15]钱婧,朱春梅,曹玲,呼吸道病原菌基因检测法诊断儿童肺炎链球菌感染的临床价值[J].中华全科医学,2018,16(9):1512-1520.
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[17]Hsieh K,Ferguson BS,Eisenstein M,et al.Integrated electrochemical microsystems for genetic detection of pathogens at the point of care[J].Acc Chem Res,2015,48(4):911-920.
[18]杜芳玲,王婷婷.环介导等温扩增(LAMP)技术的研究进展与展望[J].实验与检验医学,2018,36(4):509-512,565.
[19]张亚平,郑志高,简保磊,等.新型环介导等温扩增技术开发与应用研究进展[J].分子诊断与治疗杂志,2018,10(2):138-144.
[20]温福利.兔斯氏艾美耳球虫环介导等温扩增技术检测方法的建立[J].实验动物与比较医学,2018,38(3):176-181.
[2]Zhang C,Yao Y,Zhu JL,et al.Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms[J].Sci Rep,2016,1(6):26533.
[3]范菲楠,聂署萍,萧晓友,等.微流控技术检测宫颈脱落细胞HPV的临床应用[J].临床检验杂志:电子版,2018,7(3):375-377.
[4]黄世光,靳翔宇,林荣赞,等.微流控芯片核酸分析系统及其精准医学应用[J].中国激光,2018,45(3):124-131.
[5]王云超,牛海涛,李凯,等.微流控芯片特异性筛选膀胱癌循环肿瘤细胞的研究[J].中华泌尿外科杂志,2016,37(12):936-939.
[6]邹丽辉,李英,肖飞.微流控技术在临床检验中的应用[J].中华检验医学杂志,2018,41(9):631-633.
[7]Loo JF,Kwok HC,Leung CC,et al.Sample-to-answer on molecular diagnosis of bacterial infection using integrated lab-on-a-disc[J].Biosens Bioelectron,2017,15(93):212-219.
[8]沈韧,万谅,贾艳伟.微流控技术在临床检测中的应用[J].分子诊断与治疗杂志,2018,10(5):289-294.
[9]徐欢,陈鸣.微流控生物传感器及其分子诊断应用[J].中华检验医学杂志,2018,41(9):634-637.
[10]Zhu Z,Tang Y,Jiang YS,et al.Strand-Exchange Nucleic Acid Circuitry with Enhanced Thermo-and StructureBuffering Abilities Turns Gene Diagnostics Ultra-Reliable and Environmental Compatible[J].Sci Rep,2016,4(6):36 605.
[11]舒博文,林冬果,雷秀霞,等.微流控体外诊断技术应对临床检验医学的挑战[J].中华检验医学杂志,2018,41(9):696-699.
[12]Seyrig G,Stedtfeld RD,Tourlousse DM,et al.Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics[J].Microbiol Methods,2015,11(119):223-227.
[13]Chaumpluk P,Plubcharoensook P,Prasongsuk S.Rapid detection of aflatoxigenic Aspergillus sp.in herbal specimens by a simple,bendable,paper-based lab-on-a-chip[J]Biotechnol,2016,11(6):768-779.
[14]李松,何晓光,马可泽.环介导恒温扩增芯片法在机械通气新生儿下呼吸道病原菌检测中应用[J].广东医科大学学报,2018,36(2):169-172.
[15]钱婧,朱春梅,曹玲,呼吸道病原菌基因检测法诊断儿童肺炎链球菌感染的临床价值[J].中华全科医学,2018,16(9):1512-1520.
[16]Badolo A,Bando H,TraoréA,et al.Detection of G119Sace-1(R)mutation in field-collected Anopheles gambiae mosquitoes using allele-specific loop-mediatedisothermal amplification(AS-LAMP)method[J].Malar J,2015,14(1):477.
[17]Hsieh K,Ferguson BS,Eisenstein M,et al.Integrated electrochemical microsystems for genetic detection of pathogens at the point of care[J].Acc Chem Res,2015,48(4):911-920.
[18]杜芳玲,王婷婷.环介导等温扩增(LAMP)技术的研究进展与展望[J].实验与检验医学,2018,36(4):509-512,565.
[19]张亚平,郑志高,简保磊,等.新型环介导等温扩增技术开发与应用研究进展[J].分子诊断与治疗杂志,2018,10(2):138-144.
[20]温福利.兔斯氏艾美耳球虫环介导等温扩增技术检测方法的建立[J].实验动物与比较医学,2018,38(3):176-181.