Real-time polymerase chain reaction assays for rapid detection and virulence evaluation of the environmental Pseudomonas aeruginosa isolates
Real-time polymerase chain reaction assays for rapid detection and virulence evaluation of the environmental Pseudomonas aeruginosa isolates
复制标题
DOI：
10.1007/s11033-019-04855-y
复制
发表时间：
2019-08-01
影响因子：
2.8
通讯作者：
Douraghi, Masoumeh
中科院分区：
文献类型：
--
作者：
Golpayegani, Abdolali;Nodehi, Ramin Nabizadeh;Douraghi, Masoumeh
铜绿假单胞菌等条件致病菌的快速和特定物种检测以及毒力评估是日益引起公共卫生当局关注的问题。根据铜绿假单胞菌看家基因gyrB设计了一组引物和酶切探针，并用TaqMan定量聚合酶链式反应方法对其特异性和敏感性进行了评价。以终点聚合酶链式反应和SYBR Green定量聚合酶链式反应为对照。并以gyrB为参照，以四个毒力基因lasB、lasR、rhlR和toxA为参照，建立了多重RT-qPCRs。共采集40份环境样品、2株CF患者临床分离株、2株铜绿假单胞菌标准株和15株非靶参考菌株，检测qPCR方法的敏感性和特异性。在电子显微镜和体外跨物种实验中，证实了所设计的gyrB418F/gyrB490R/gyrB444P具有高的特异性和低的跨物种扩增。TaqMan和SYBR Green qPCRs对所有目标铜绿假单胞菌的敏感度均为100%，检测到的细菌数量低于10个基因组当量。基因稳定性测定的最低M值为0.19，证明gyrB基因适合作为RT-qPCR的参考基因。所开发的QPCRS具有足够的检测能力来鉴定环境样品中的铜绿假单胞菌，包括清洁和娱乐用水、经处理和未经处理的污水和土壤。我们设计的引物和探针的扩增片段长度较短，加上M值较低，因此是一种适合于RT-qPCR检测毒力基因表达的方法。
Rapid and species-specific detection, and virulence evaluation of opportunistic pathogens such as Pseudomonas aeruginosa, are issues that increasingly has attracted the attention of public health authorities. A set of primers and hydrolysis probe was designed based on one of the P. aeruginosa housekeeping genes, gyrB, and its specificity and sensitivity was evaluated by TaqMan qPCR methods. The end point PCR and SYBR Green qPCR were used as control methods. Furthermore, multiplex RT-qPCRs were developed for gyrB as reference and four virulence genes, including lasB, lasR, rhlR and toxA. Totally, 40 environmental samples, two clinical isolates from CF patients, two standard strains of P. aeruginosa, and 15 non-target reference strains were used to test the sensitivity and specificity of qPCR assays. In silico and in vitro cross-species testing confirmed the high specificity and low cross-species amplification of the designed gyrB418F/gyrB490R/gyrB444P. The sensitivity of both TaqMan and SYBR Green qPCRs was 100% for all target P. aeruginosa, and the detected count of bacteria was below ten genomic equivalents. The lowest M value obtained from gene-stability measurement was 0.19 that confirmed the suitability of gyrB as the reference gene for RT-qPCR. The developed qPCRs have enough detection power for identification of P. aeruginosa in environmental samples including clean and recreational water, treated and untreated sewage and soil. The short amplicon length of our designed primers and probes, alongside with a low M value, make it as a proper methodology for RT-qPCR in virulence genes expression assessment.