PCR PRIMERS AND PROBES FOR THE 16S RIBOSOMAL-RNA GENE OF MOST SPECIES OF PATHOGENIC BACTERIA, INCLUDING BACTERIA FOUND IN CEREBROSPINAL-FLUID
PCR PRIMERS AND PROBES FOR THE 16S RIBOSOMAL-RNA GENE OF MOST SPECIES OF PATHOGENIC BACTERIA, INCLUDING BACTERIA FOUND IN CEREBROSPINAL-FLUID
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DOI：
10.1128/jcm.32.2.335-351.1994
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发表时间：
1994-02-01
影响因子：
9.4
通讯作者：
LEONG, D
中科院分区：
文献类型：
--
作者：
GREISEN, K;LOEFFELHOLZ, M;LEONG, D
检测了一组针对细菌中16S rRNA基因的广谱聚合酶链式反应(16S RRNA)引物，以及三个系列的寡核苷酸探针来检测PCR产物。第一个系列的探针范围很广，包括一个通用细菌探针，一个革兰氏阳性探针，一个类杆菌-黄杆菌探针，以及两个针对其他革兰氏阴性物种的探针。第二个系列的目的是检测经常引起脑膜炎的七个主要细菌种或组的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、单核细胞增生性李斯特菌和金黄色葡萄球菌。第三个系列用于检测通常被认为是临床样本潜在污染物的物种或属的DNA，包括脑脊液(CSF)：芽孢杆菌、棒状杆菌、丙酸杆菌和凝固酶阴性葡萄球菌。这些引物扩增了所测试的所有124种不同细菌的DNA。用含有3M四甲基氯化铵的洗涤剂对宽范围探针进行Southern杂交测试表明，这组探针正确识别了102种细菌中除两种外的所有细菌，例外是放射性葡萄球菌和阴道加德纳氏菌。革兰氏阴性和革兰氏阳性探针与革兰氏染色特征预测的两种新特征细菌--异种球菌性中耳炎和亨氏罗氏杆菌的分离株杂交。将脑脊液病原体和污染物探针序列与现有的序列信息和32个不同物种的测序数据进行了比较。对来自60多个不同菌株的脑脊液病原体和污染探针的DNA检测表明，除了凝固酶阴性的葡萄球菌探针外，这些探针可以正确地鉴定已知在脑脊液中发现的细菌种类。
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.