Chen Ying, Sha Lulu, Li Wenqing, Zhou Liuyan, Pei Bing, Bian Xinyu, Ji Yongxin, Liu Yiping, Wang Li, Yang Huan
School of Medical Technology, Xuzhou Medical University, Xuzhou, China.
Department of Clinical Laboratory, Suqian First People's Hospital, Suqian, China.
Front Bioeng Biotechnol. 2023 Feb 8;11:1144463. doi: 10.3389/fbioe.2023.1144463. eCollection 2023.
(KP, ) is one of the most important nosocomial pathogens that cause severe respiratory infections. As evolutionary high-toxic strains with drug resistance genes increase year by year, the infections caused by it are often accompanied by high mortality, which may be fatal to infants and can cause invasive infections in healthy adults. At present, the traditional clinical methods for detecting are cumbersome and time-consuming, and the accuracy and sensitivity are not high. In this study, nanofluorescent microsphere (nFM)-based immunochromatographic test strip (ICTS) quantitative testing platform were developed for point-of-care testing (POCT) method of . 19 clinical samples of infants were collected, the genus-specific gene of was screened from Polymerase chain reaction (PCR) combined with nFM-ICTS based on magnetic purification assay (PCR-ICTS) and strand exchange amplification (SEA) combined with nFM-ICTS based on magnetic purification assay (SEA-ICTS) were developed for the quantitative detection of The sensitivity and specificity of SEA-ICTS and PCR-ICTS were demonstrated by the existing used classical microbiological methods, the real-time fluorescent quantitative PCR (RTFQ-PCR) and PCR assay based on agarose gel electrophoresis (PCR-GE). Under optimum working conditions, the detection limits of PCR-GE, RTFQ-PCR, PCR-ICTS and SEA-ICTS are 7.7 × 10, 2.5 × 10, 7.7 × 10, 2.82 × 10 ng/μL, respectively. The SEA-ICTS and PCR-ICTS assays can quickly identify , and could specifically distinguish samples from non- samples. Experiments have shown a diagnostic agreement of 100% between immunochromatographic test strip methods and the traditional clinical methods on the detection of clinical samples. During the purification process, the Silicon coated magnetic nanoparticles (Si-MNPs) were used to removed false positive results effectively from the products, which showed of great screening ability. The SEA-ICTS method was developed based on PCR-ICTS, which is a more rapid (20 min), low-costed method compared with PCR-ICTS assay for the detection of in infants. Only need a cheap thermostatic water bath and takes a short detection time, this new method can potentially serve as an efficient point-of-care testing method for on-site detection of pathogens and disease outbreaks without fluorescent polymerase chain reaction instruments and professional technicians operation.
肺炎克雷伯菌（KP）是引起严重呼吸道感染的最重要的医院病原体之一。随着携带耐药基因的高毒性进化菌株逐年增加，由其引起的感染往往伴随着高死亡率，这对婴儿可能是致命的，并且可在健康成年人中引起侵袭性感染。目前，传统的临床检测方法繁琐且耗时，准确性和灵敏度不高。在本研究中，开发了基于纳米荧光微球（nFM）的免疫层析试纸条（ICTS）定量检测平台，用于肺炎克雷伯菌的即时检测（POCT）方法。收集了19份婴儿临床样本，通过聚合酶链反应（PCR）结合基于磁性纯化分析的nFM-ICTS（PCR-ICTS）和链置换扩增（SEA）结合基于磁性纯化分析的nFM-ICTS（SEA-ICTS）从样本中筛选肺炎克雷伯菌的属特异性基因，用于肺炎克雷伯菌的定量检测。通过现有的经典微生物学方法、实时荧光定量PCR（RTFQ-PCR）和基于琼脂糖凝胶电泳的PCR检测（PCR-GE）证明了SEA-ICTS和PCR-ICTS的灵敏度和特异性。在最佳工作条件下，PCR-GE、RTFQ-PCR、PCR-ICTS和SEA-ICTS的检测限分别为7.7×10、2.5×10、7.7×10、2.82×10 ng/μL。SEA-ICTS和PCR-ICTS检测方法可以快速鉴定肺炎克雷伯菌，并能特异性地区分肺炎克雷伯菌样本和非肺炎克雷伯菌样本。实验表明，免疫层析试纸条方法与传统临床方法在临床样本检测上的诊断一致性为100%。在纯化过程中，使用硅包被的磁性纳米颗粒（Si-MNPs）有效去除产物中的假阳性结果，显示出很强的筛选能力。SEA-ICTS方法是在PCR-ICTS的基础上开发的，与PCR-ICTS检测婴儿肺炎克雷伯菌的方法相比，它是一种更快速（20分钟）、低成本的方法。这种新方法只需要一个便宜的恒温水浴，检测时间短，在没有荧光聚合酶链反应仪器和专业技术人员操作的情况下，有可能作为一种有效的即时检测方法用于现场病原体检测和疾病爆发检测。
