Selvapandiyan Angamuthu, Stabler Katie, Ansari Nasim A, Kerby Stephen, Riemenschneider Jenny, Salotra Poonam, Duncan Robert, Nakhasi Hira L
Division of Emerging and Transfusion Transmitted Disease, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
J Mol Diagn. 2005 May;7(2):268-75. doi: 10.1016/S1525-1578(10)60554-5.
A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.
利用赛沛智能循环仪平台开发了一种多重聚合酶链反应检测方法，用于快速同时检测血液中的A类特定细菌病原体（炭疽芽孢杆菌和鼠疫耶尔森菌）和寄生虫病原体（利什曼原虫属）。分别使用炭疽芽孢杆菌（斯特恩株）和假结核耶尔森菌对该检测方法进行优化，以检测炭疽芽孢杆菌和鼠疫耶尔森菌。通过用SYBR Green I对扩增子进行染色并测定其各自的解链温度（Tm），评估了目标扩增子[炭疽芽孢杆菌的保护性抗原基因以及其他病原体或人类的rRNA基因（内对照）]的特异性。作为病原体半定量的一种新方法，扩增子的Tm峰高与已知的病原体加标样品标准曲线相关。该检测方法能够检测出每毫升血液中加标少于50个靶细胞的所有病原体的DNA。该检测方法对利什曼病患者血液中杜氏利什曼原虫和有症状小鼠血液中炭疽芽孢杆菌（斯特恩株）的检测灵敏度为100%。包括样品制备在内，进行该检测所需的时间少于1.5小时，这使其成为一种潜在的有用方法，可用于快速诊断和监测感染个体中药物或疫苗的疗效。
