Rattanathongkom A, Sermswan R W, Wongratanacheewin S
Department of Oral Biology, Faculty of Dentistry, Khon Kaen University, Thailand.
Mol Cell Probes. 1997 Feb;11(1):25-31. doi: 10.1006/mcpr.1996.0072.
A highly sensitive, specific, rapid and simple method to detect Burkholderia pseudomallei in blood samples was developed. Two 22-base oligonucleotide primers, based on sequences from a specific DNA probe, were used for amplification of bacterial DNA by the polymerase chain reaction (PCR). Amplification with these primers yielded a 178-base pair product in 100 clinical isolates of B. pseudomallei. As little as 0.5 fg of B. pseudomallei DNA was detectable by this method. Experiments involving inoculation of the organism into uninfected blood samples showed that the method could be used to detect as few as 1 bacterial cell ml-1 of whole blood. Non-specific amplification of other bacterial DNAs from 18 samples of bacteria was not observed. Blood samples from seven patients proven to have melioidosis by haemoculture were positive using these primers. The total time required for sample processing, amplification and visualization was approximately 3.5 h. The high sensitivity, rapidity and simplicity of this method should make it valuable for diagnosis, monitoring of drug treatment and for epidemiological studies of the melioidosis.
开发了一种用于检测血液样本中类鼻疽伯克霍尔德菌的高灵敏度、特异性、快速且简便的方法。基于特定DNA探针的序列设计了两条22个碱基的寡核苷酸引物，用于通过聚合酶链反应（PCR）扩增细菌DNA。用这些引物对100株类鼻疽伯克霍尔德菌临床分离株进行扩增，产生了一个178碱基对的产物。该方法可检测低至0.5 fg的类鼻疽伯克霍尔德菌DNA。将该菌接种到未感染血液样本中的实验表明，该方法可用于检测全血中低至每毫升1个细菌细胞。未观察到来自18种细菌样本的其他细菌DNA的非特异性扩增。使用这些引物对7例经血培养证实患有类鼻疽病的患者的血液样本检测呈阳性。样本处理、扩增和可视化所需的总时间约为3.5小时。该方法的高灵敏度、快速性和简便性使其在类鼻疽病的诊断、药物治疗监测和流行病学研究中具有重要价值。
