Greisen K, Loeffelholz M, Purohit A, Leong D
Roche Molecular Systems, Alameda, California 94501.
J Clin Microbiol. 1994 Feb;32(2):335-51. doi: 10.1128/jcm.32.2.335-351.1994.
A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.
对一组用于细菌16S rRNA基因的广谱PCR引物进行了测试，并使用了三个系列的寡核苷酸探针来检测PCR产物。第一系列探针范围广泛，包括通用细菌探针、革兰氏阳性探针、拟杆菌-黄杆菌探针以及针对其他革兰氏阴性菌的两个探针。第二系列旨在检测七种常见引起脑膜炎的主要细菌物种或菌群的PCR产物：脑膜炎奈瑟菌、流感嗜血杆菌、肺炎链球菌、无乳链球菌、大肠杆菌和其他肠道细菌、单核细胞增生李斯特菌以及金黄色葡萄球菌。第三系列旨在检测通常被认为是临床样本（包括脑脊液，CSF）潜在污染物的物种或属的DNA：芽孢杆菌属、棒状杆菌属、丙酸杆菌属以及凝固酶阴性葡萄球菌属。这些引物扩增了所测试的所有124种不同细菌的DNA。用含3M四甲基氯化铵的洗液对广谱探针进行Southern杂交检测表明，这组探针正确鉴定了所测试的102种细菌中的100种，例外的是耐辐射异常球菌和阴道加德纳菌。革兰氏阴性和革兰氏阳性探针与两种新鉴定的细菌——中耳炎差异球菌和汉氏罗卡利马氏体的分离株杂交，正如革兰氏染色特征所预测的那样。将CSF病原体和污染物探针序列与可用序列信息以及32种不同物种的测序数据进行了比较。用超过60种不同菌株的DNA对CSF病原体和污染物探针进行测试表明，除凝固酶阴性葡萄球菌探针外，这些探针能够正确鉴定已知存在于CSF中的细菌物种。
