Clancy Eoin, Higgins Owen, Forrest Matthew S, Boo Teck Wee, Cormican Martin, Barry Thomas, Piepenburg Olaf, Smith Terry J
Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland.
Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
BMC Infect Dis. 2015 Oct 29;15:481. doi: 10.1186/s12879-015-1212-5.
Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.
Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.
The RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive.
The RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.
肺炎链球菌是人类微生物疾病的重要病因。多价疫苗的引入与肺炎球菌相关死亡人数的急剧下降同时发生。尽管如此，在全球范围内，肺炎球菌感染仍然是5岁以下儿童和65岁及以上成年人死亡的重要原因。为了妥善管理患者并控制感染传播，需要一种快速且高度灵敏的诊断方法用于常规检测，尤其是在肺炎球菌疾病最为普遍的资源有限地区。
以编码前导肽酶A的基因作为分子诊断靶点，设计并优化了一种实时RPA检测方法，用于检测全血中的肺炎链球菌。在分析检测限和特异性方面，将该检测方法与实时PCR进行了比较。研究了人类基因组DNA对扩增的抑制作用。使用少量临床样本研究了该检测方法的潜在临床应用价值。
RPA检测方法的检测限与PCR相当（分别为每个反应4.0和5.1个基因组当量），当接种到人类全血中时，能够检测到相当于<1个肺炎链球菌菌落形成单位的量。RPA检测方法的包容性为100%（38/38个实验室参考菌株和19/19个侵袭性临床分离株），排他性为100%；能够区分肺炎链球菌菌株与其他草绿色链球菌，包括伪肺炎链球菌。当应用于对少量（n = 11）临床样本（肺炎链球菌血培养阳性）的分析时，RPA检测方法被证明既快速又灵敏。
本研究中开发的RPA检测方法与PCR一样灵敏和特异。在反应动力学方面，RPA检测方法优于PCR，RPA在20分钟内完成反应，最短只需6分钟就能产生阳性信号。这项研究代表了一种可能适用于即时检测环境的检测方法。
