Sheppard Carmen L, Harrison Timothy G, Morris Rhonwen, Hogan Angela, George Robert C
Respiratory and Systemic Infection Laboratory (RSIL), Health Protection Agency (HPA) Central Public Health Laboratory (CPHL), 61 Colindale Avenue, London NW9 5HT, UK 2Vaccine Evaluation Unit, Microbiology Department, Gloucestershire Royal Hospital, Gloucester GL1 3NN, UK.
J Med Microbiol. 2004 Mar;53(Pt 3):189-195. doi: 10.1099/jmm.0.05460-0.
The development and clinical evaluation of a LightCycler PCR assay, including an internal process control (IPC), to detect the Streptococcus pneumoniae autolysin gene in clinical samples is reported. The assay was developed to provide a second target for use in conjunction with existing pneumolysin PCR assays to increase the reliability of non-culture PCR diagnosis of pneumococcal infection. Primers amplify a 173 bp fragment of the autolysin gene (lytA), which is detected by fluorescence-labelled hybridization probes. An IPC was designed to check for the presence of PCR inhibitors and loss of assay sensitivity. The IPC product was amplified by the lytA primers and detected by a second set of hybridization probes. The analytical specificity of the autolysin PCR assay was 100% against 39 other bacterial species tested; these included related streptococci and other organisms. The assay, which could reliably detect 50 fg purified pneumococcal DNA per reaction, was capable of distinguishing between S. pneumoniae and atypical Streptococcus mitis and Streptococcus oralis strains known to contain the lytA gene. Using DNA extracts from a panel of EDTA bloods from patients with blood-culture-confirmed pneumococcal infection, the autolysin PCR had a sensitivity of 42.9%, which was similar to a previously reported TaqMan pneumolysin PCR (43.8%) run in parallel. Total agreement was shown between the autolysin assay and the pneumolysin TaqMan assay when used to test 23 culture-negative clinical samples, of which eight were positive by PCR, adding valuable clinical information. A specific autolysin-based LightCycler assay has been developed to complement pneumolysin PCR for the detection of S. pneumoniae in clinical samples. This should be a particularly useful tool for the rapid and sensitive diagnosis of pneumococcal meningitis, even after an antibiotic has been administered. However, poor sensitivity on blood samples limits its usefulness in other bacteraemic infections.
本文报道了一种用于检测临床样本中肺炎链球菌自溶素基因的LightCycler PCR检测方法的开发及临床评估，该方法包括一个内部过程控制（IPC）。开发此检测方法的目的是提供第二个靶点，与现有的肺炎溶血素PCR检测方法联合使用，以提高肺炎球菌感染非培养PCR诊断的可靠性。引物扩增自溶素基因（lytA）的173 bp片段，该片段通过荧光标记的杂交探针进行检测。设计了一个IPC来检查PCR抑制剂的存在以及检测方法敏感性的丧失。IPC产物由lytA引物扩增，并由第二组杂交探针检测。自溶素PCR检测方法对39种其他受试细菌的分析特异性为100%；这些细菌包括相关链球菌和其他微生物。该检测方法每个反应能够可靠地检测到50 fg纯化的肺炎球菌DNA，能够区分肺炎链球菌与已知含有lytA基因的非典型缓症链球菌和口腔链球菌菌株。使用来自血培养确诊的肺炎球菌感染患者的一组EDTA血液的DNA提取物，自溶素PCR的敏感性为42.9%，与同时进行的先前报道的TaqMan肺炎溶血素PCR（43.8%）相似。在用于检测23份培养阴性的临床样本时，自溶素检测方法与肺炎溶血素TaqMan检测方法完全一致，其中8份样本通过PCR呈阳性，增加了有价值的临床信息。已开发出一种基于自溶素的特异性LightCycler检测方法，以补充肺炎溶血素PCR用于临床样本中肺炎链球菌的检测。这对于肺炎球菌性脑膜炎的快速和灵敏诊断应该是一个特别有用的工具，即使在使用抗生素后也是如此。然而，对血液样本的低敏感性限制了其在其他菌血症感染中的应用。
