Rapid label-free identification of mixed bacterial infections by surface plasmon resonance
Rapid label-free identification of mixed bacterial infections by surface plasmon resonance
复制标题
通过表面等离子体共振快速无标记鉴定混合细菌感染
DOI：
10.1186/1740-3391-9-11
复制
发表时间：
2011-06-07
影响因子：
7.4
通讯作者：
Liao, Pu
中科院分区：
文献类型：
--
作者：
Wang, Jue;Luo, Yang;Liao, Pu
背景：由于复杂环境中的表型变化，需氧和厌氧混合感染的早期检测在临床实践中一直是一个挑战。表面等离子体共振(SPR)生物传感器被广泛应用于DNA-DNA相互作用的检测，为DNA研究提供了一种灵敏、无标记的方法。方法：本研究建立了单链DNA(SsDNA)扩增技术，并对传统的SPR检测系统进行了改进，以快速同时检测4种病原微生物(铜绿假单胞菌、金黄色葡萄球菌、破伤风杆菌和产气荚膜梭菌)的混合感染。结果：我们构建了良好的循环检测系统，提高了灵敏度，并改进了串联探针阵列，减少了非特异性杂交。16S rDNA通用引物的使用确保了4个目的核酸序列的同时扩增，进一步的电泳和测序证实了该扩增方法的高效性。在单碱基错配或非特异性探针杂交过程中未检测到显著信号(P<；0.05)。四种细菌扩增产物的校正曲线在0.1~100 nm范围内线性关系良好，R(2)值均为0.99。最低检测限铜绿假单胞菌0.03 nM、金黄色葡萄球菌0.02 nM、破伤风杆菌0.01 nM、产气荚膜梭菌0.02 nM。SPR生物传感器与传统培养法具有相同的检测率(P<；0.05)。结论：本方法能快速、准确地鉴定需氧和厌氧混合感染，为细菌培养的快速检测提供了可靠的替代方法。
Background: Early detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Surface plasmon resonance (SPR) biosensor is widely used to detect DNA-DNA interaction and offers a sensitive and label-free approach in DNA research.Methods: In this study, we developed a single-stranded DNA (ssDNA) amplification technique and modified the traditional SPR detection system for rapid and simultaneous detection of mixed infections of four pathogenic microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium tetani and Clostridium perfringens).Results: We constructed the circulation detection well to increase the sensitivity and the tandem probe arrays to reduce the non-specific hybridization. The use of 16S rDNA universal primers ensured the amplification of four target nucleic acid sequences simultaneously, and further electrophoresis and sequencing confirmed the high efficiency of this amplification method. No significant signals were detected during the single-base mismatch or non-specific probe hybridization (P < 0.05). The calibration curves of amplification products of four bacteria had good linearity from 0.1 nM to 100 nM, with all R(2) values of > 0.99. The lowest detection limits were 0.03 nM for P. aeruginosa, 0.02 nM for S. aureus, 0.01 nM for C. tetani and 0.02 nM for C. perfringens. The SPR biosensor had the same detection rate as the traditional culture method (P < 0.05). In addition, the quantification of PCR products can be completed within 15 min, and excellent regeneration greatly reduces the cost for detection.Conclusions: Our method can rapidly and accurately identify the mixed aerobic-anaerobic infection, providing a reliable alternative to bacterial culture for rapid bacteria detection.