背景:本研究旨在建立一种通过使用多酶等温快速扩增(MIRA)技术快速检测高毒力肺炎克雷伯菌(hvKP)的方法。实验室可以迅速,准确地说,并方便地诊断高毒力肺炎克雷伯菌感染。
方法:对于本研究,收集7株实验室标准菌株和184株临床分离株(其中肺炎克雷伯菌70株),根据其菌落形态筛选高毒力肺炎克雷伯菌,拉丝试验,和下一代测序(NGS)结果。基于GenBank上高毒力肺炎克雷伯菌peg344基因的核酸序列(编号:AP006726.1),选择特异性保守区设计MIRA和实时荧光定量PCR(qPCR)特异性引物和探针。采用MIRA和qPCR方法检测受试菌株,和特异性,灵敏度,并评估了MIRA方法检测hvKP的临床性能。
结果:总计,从临床分离株中筛选出21例hvKP。MIRA检测方法利用特异性引物和探针在39°C下传输显著的荧光信号,检测过程需要30分钟。特异性检测结果显示,只有hvKP具有特异性扩增曲线,而其余非高毒力肺炎克雷伯菌(non-hvKP)无特异性扩增曲线。灵敏度测试结果表明,MIRA检测hvKP的灵敏度为7×102CFU/mL,这与实时荧光qPCR方法的灵敏度是一致的。通过使用MIRA和qPCR方法同时检测184个临床分离株。21株hvKP具有特异性扩增曲线,而其余163株非hvKP无特异性扩增曲线。检测hvKP的两种方法的准确度均为100%。
结论:建立的多酶等温快速扩增(MIRA)具有以下特点:检测时间短,高灵敏度,和强烈的特异性,可作为hvKp早期诊断和流行病学监测的有力工具。
BACKGROUND: This study aimed to establish a method for the rapid detection of highly virulent Klebsiella pneumoniae (hvKP) by using multienzyme isothermal rapid amplification (MIRA) technology. The laboratory can quickly, accurately, and conveniently diagnose highly virulent Klebsiella pneumoniae infection.
METHODS: For this study, 7 laboratory standard strains and 184 clinical isolates (including 70 strains of Klebsiella pneumoniae) were collected and screened for highly virulent Klebsiella pneumoniae based on its colony morphology, wire drawing test, and next-generation sequencing (NGS) results. Based on the nucleic acid sequence of the peg344 gene of highly virulent Klebsiella pneumoniae on GenBank (no. AP006726.1), specific conserved regions were selected to design MIRA and real-time fluorescence quantitative PCR (qPCR) specific primers and probes. The MIRA and qPCR methods were used to detect the tested strain, and the specificity, sensitivity, and clinical performance of the MIRA method for detecting hvKP were evaluated.
RESULTS: In total, 21 cases of hvKP were screened from clinical isolates. The MIRA detection method utilizes specific primers and probes to transmit significant fluorescence signals at 39°C, and the detection process takes 30 minutes. The specificity test results showed that only hvKP had a specific amplification curve, while the rest of non-highly virulent Klebsiella pneumoniae (non-hvKP) had no specific amplification curve. The sensitivity test results showed that the sensitivity of MIRA for detecting hvKP is 7 × 102 CFU/mL, which is consistent with the sensitivity of the real-time fluorescence qPCR method. A simultaneous detection of 184 clinical isolates was accomplished by using MIRA and qPCR methods. Twenty-one strains of hvKP have specific amplification curves, while the remaining 163 strains of non-hvKP have no specific amplification curves. The accuracy of both methods for detecting hvKP is 100%.
CONCLUSIONS: The established multienzyme isothermal rapid amplification (MIRA) has the following characteristics: a short detection time, high sensitivity, and a strong specificity, and it can be used as a powerful tool for an early diagnosis and epidemiological monitoring of hvKp.