Kim So Yeon, Yun Young-Sun, Lee Kwang-Jun, Kim Jonghyun
Division of Zoonotic and Vector-borne Disease Research, Center for Infectious Diseases Research, Korea National Institute of Health, Cheongju, South Korea.
Division of Acute Viral Disease, Center for Emerging Virus Research, Korea National Institute of Health, Cheongju, South Korea.
Microbiol Spectr. 2025 Apr;13(4):e0190724. doi: 10.1128/spectrum.01907-24. Epub 2025 Feb 18.
This study describes a method for detecting in patient stools with subsequent isolation using antibody-magnetic beads in conjunction with selective culture and PCR. Monoclonal antibodies specific for the flagellin A and major outer membrane protein of were generated; two clones (1C7 and 4B2) were used to coat magnetic beads for immunomagnetic separation (IMS). strain NCTC11168 was recovered from human stool samples spiked with varying concentrations (10-10 CFU/mL) by (Campy)-IMS or a conventional culture-based method and plated on modified charcoal-cefoperazone-deoxycholate agar; the number of colonies was enumerated. The detection limits of Campy-IMS and conventional culture-based method with spiked stool samples were 10 and 10 CFU/mL, respectively. The sensitivity of IMS-PCR was 10-10,000-fold higher than that of direct PCR. The recovery rate of from spiked stools stored for 12 to 72 h decreased from 72.3 to 5.9% with Campy-IMS and from 48.5 to 0.1% with the conventional culture-based method. Importantly, of 20 PCR (+)/bacterial culture (-) samples that were diagnosed as probable cases according to general criteria, 95% (19/20) were confirmed positive by Campy-IMS. Thus, this study suggests a solution to overcome the problems caused by the inconsistency between probable and confirmed cases of infection.
The isolation, cultivation, and maintenance of spp. are difficult because of the microaerophilic conditions and specific medium needed. Although selective media are useful for the initial isolation of , subsequent exposure of the sample to oxygen has a detrimental effect on the positive culture rate of , significantly lowering the isolation rate from patient samples. In this study, the detection limit was improved by combining immunomagnetic separation and PCR methods to quickly detect in clinical patient stool samples using antibodymagnetic beads. Therefore, this study is expected to improve confirmation of infection where diagnosis would previously fail with patient samples because of oxygen exposure, inappropriate diagnostic methods, and interference from other bacteria in the sample.
本研究描述了一种在患者粪便中进行检测的方法，随后使用抗体磁珠结合选择性培养和聚合酶链反应（PCR）进行分离。制备了针对空肠弯曲菌鞭毛蛋白A和主要外膜蛋白的单克隆抗体；使用两个克隆（1C7和4B2）包被磁珠用于免疫磁分离（IMS）。通过弯曲菌免疫磁分离（Campy-IMS）或传统的基于培养的方法，从添加了不同浓度（10⁻¹⁰CFU/mL）的人粪便样本中回收空肠弯曲菌NCTC11168菌株，并接种于改良的炭头孢哌酮脱氧胆酸盐琼脂平板上；计数菌落数量。添加粪便样本的Campy-IMS和传统基于培养方法的检测限分别为10⁻¹⁰和10⁻⁹CFU/mL。IMS-PCR的灵敏度比直接PCR高10⁻¹⁰,000倍。使用Campy-IMS，添加的粪便样本储存12至72小时后，空肠弯曲菌的回收率从72.3%降至5.9%，而使用传统基于培养的方法，回收率从48.5%降至0.1%。重要的是，根据一般标准被诊断为可能病例的20份PCR（+）/细菌培养（-）样本中，95%（19/20）通过Campy-IMS确认为阳性。因此，本研究提出了一种解决方案，以克服空肠弯曲菌感染可能病例与确诊病例之间不一致所导致的问题。
由于需要微需氧条件和特定培养基，空肠弯曲菌属的分离、培养和保存较为困难。虽然选择性培养基有助于空肠弯曲菌的初步分离，但随后样本暴露于氧气会对空肠弯曲菌的阳性培养率产生不利影响，显著降低患者样本的分离率。在本研究中，通过结合免疫磁分离和PCR方法，使用抗体磁珠快速检测临床患者粪便样本中的空肠弯曲菌，提高了检测限。因此，本研究有望改善空肠弯曲菌感染的确诊情况，此前由于氧气暴露、诊断方法不当以及样本中其他细菌的干扰，患者样本的诊断可能会失败。
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