Kunthalert Duangkamol, Henghiranyawong Kritsada, Sistayanarain Anchalee, Khoothiam Krissana
Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand.
Int J Pediatr Otorhinolaryngol. 2013 Feb;77(2):275-80. doi: 10.1016/j.ijporl.2012.11.019. Epub 2012 Dec 13.
The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance.
We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates.
The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%.
Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms.
全球细菌性中耳炎的极高患病率促使人们进行适当的疾病管理。虽然中耳炎疫苗的研发前景广阔，但可靠有效的此类病原体诊断方法至关重要。
我们开发了一种多重聚合酶链反应检测方法，用于同时检测和区分不可分型和可分型的流感嗜血杆菌、卡他莫拉菌和肺炎链球菌。在这一单步聚合酶链反应中纳入了五对引物，分别靶向延胡索酸还原酶基因（流感嗜血杆菌）、外膜蛋白B基因（卡他莫拉菌）、主要自溶素基因（肺炎链球菌）、与荚膜形成相关的BexA蛋白基因（所有有荚膜的流感嗜血杆菌）和16S rRNA基因。还对临床分离株进行了多重聚合酶链反应的验证。
所开发的多重聚合酶链反应具有高度特异性，能够以特定方式检测目标病原体，无论是单独检测还是作为所有目标生物体的混合物检测。该检测方法也很灵敏，最低检测限为1纳克细菌DNA。当应用于来自不同标本来源的临床分离株时，本研究开发的多重聚合酶链反应能够正确地单独鉴定每种微生物，或鉴定两种或更多目标生物体的组合。所有结果均与传统培养鉴定结果相符。此外，该检测方法区分本研究临床分离株中流感嗜血杆菌荚膜形成的能力为100%。
我们的多重聚合酶链反应为检测这4种目标生物体提供了一种快速准确的诊断工具。这种检测方法将成为临床医生和流行病学家对由这些生物体引起的疾病进行适当治疗和管理的有用工具。
