【摘要】 目的 筛选多交叉置换扩增技术（MCDA）结合纳米生物传感条（LFB）检测肺炎克雷伯菌的最佳扩增温度、最佳扩增时间，并对MCDA-LFB法检测肺炎克雷伯菌的效果进行评估。方法 根据肺炎克雷伯菌特异的保守基因rcsA的核苷酸序列，采用Primer Premier Version 5.0软件设计一套特异性MCDA引物，其中交叉引物CP1和扩增引物C1分别标记异硫氰酸荧光素（FITC）和地高辛（Dig），建立25μL的MCDA反应体系。将温度按60℃、61℃、62℃、63℃、64℃、65℃、66℃、67℃依次递增，分别扩增1 h，然后85℃加热5 min后终止反应，使用实时浊度仪观察DNA浓度曲线，绘制MCDA扩增反应动力学图，确定最佳扩增温度。在筛选出的最佳扩增温度下，选择10 min、20 min、30 min和40 min 4个时间点分别对10 ng/μL、100 pg/μL、10 pg/μL、1 pg/μL、100 fg/μL、10 fg/μL和1 fg/μL的肺炎克雷伯菌DNA进行MCDA扩增，使用LFB进行检测，根据LFB检测结果确定最佳扩增时间。使用MCDA-LFB法检测36株肺炎克雷伯菌和18株非肺炎克雷伯菌，使用临床分离培养法和MCDA-LFB法检测呼吸道感染患者的42份痰液样本和3份健康对照痰液样本，观察MCDA-LFB法检测肺炎克雷伯菌的灵敏度、特异度。将肺炎克雷伯菌ATCC700603标准株基因组DNA分别稀释至10ng/μL、100pg/μL、10pg/μL、1pg/μL、100 fg/μL、10 fg/μL和1 fg/μL，使用MCDA-LFB法检测，依据结果是否阳性判定检测下限。结果 MCDA-LFB检测肺炎克雷伯菌的最佳扩增温度为63℃，最佳扩增时间为30 min。MCDA-LFB法检测肺炎克雷伯菌的灵敏度、特异度均为100%。MCDA-LFB检测肺炎克雷伯菌的检测下限为100 fg/μL。结论 MCDA-LFB检测肺炎克雷伯菌的最佳扩增温度为63℃，最佳扩增时间为30 min。MCDA-LFB检测肺炎克雷伯菌的方法简单、快速、特异、灵敏，可以用于临床呼吸道标本中的肺炎克雷伯菌早期筛选。更多还原

【Abstract】 Objective To select the best amplification temperature and time for detection of Klebsiella pneumoniae by multiple cross displacement amplification(MCDA) combined with nanoparticles-based lateral flow biosensor(LFB), and to evaluate the effect of MCDA-LFB method for detection of Klebsiella pneumoniae. Methods According to the nucleotide sequence of the specific conserved gene rcsA of Klebsiella pneumoniae, a set of specific MCDA primers were designed using Primer Premier Version 5. 0 software, in which the crossover primer CP1 and the amplification primer C1were labeled with fluorescein isothiocyanate(FITC) and digoxin(Dig), respectively, to establish 25 μL MCDA reaction system. The temperature increased by 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃ and 67 ℃, each amplification for 1 h, respectively, and then the reaction was terminated after heating at 85 ℃ for 5 minutes. The DNA concentration curve was observed by real-time turbidimeter, and the MCDA amplification reaction kinetics diagram was drawn to determine the optimal amplification temperature. Under the selected optimum amplification temperature, MCDA of 10 ng/μL, 100 pg/μL, 10 pg/μL, 1 pg/μL, 100 fg/μL, 10 fg/μL, and 1 fg/μL of Klebsiella pneumoniae DNA was amplified at four time points of 10, 20, 30 and 40 min, and LFB was used for detection. The best amplification time was determined according to the LFB detection results. Thirty-six strains of Klebsiella pneumoniae and 18 strains of non-Klebsiella pneumoniae were detected by MCDA-LFB method, 42 sputum samples from patients with respiratory tract infection and 3 healthy control sputum samples were detected by clinical isolation and culture method and MCDA-LFB method, and the sensitivity and specificity of MCDA-LFB method in detecting Klebsiella pneumoniae were observed. We diluted the genomic DNA of Klebsiella pneumoniae ATCC700603 standard strain to 10 ng/μL, 100 pg/μL, 10 pg/μL, 1 pg/μL, 100fg/μL, 10 fg/μL, and 1 fg/μL, respectively, and used MCDA-LFB method to determine the lower limit of detection based on whether the result was positive. Results The optimal amplification temperature and time for detection of Klebsiella pneumoniae by MCDA-LFB were 63 ℃ and 30 min. The sensitivity and specificity of MCDA-LFB method for detecting Klebsiella pneumoniae were 100%. The lower limit of detection of Klebsiella pneumoniae by MCDA-LFB was 100 fg/μL.Conclusions The optimal amplification temperature and time for detection of Klebsiella pneumoniae by MCDA-LFB are 63 ℃ and 30 min. The method of detecting Klebsiella pneumoniae by MCDA-LFB is simple, rapid, specific and sensitive, and can be used for early screening of Klebsiella pneumoniae in clinical respiratory tract samples.更多还原