血液中肺炎链球菌的AFLP快速检测方法的建立(1)
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[摘要] 目的 建立扩增片段长度多态性(AFLP)快速检测血液中肺炎链球菌的方法。方法 针对肺炎链球菌的管家基因16SrRNA基因设计特异引物，优化反应条件，建立肺炎链球菌的AFLP检测方法。通过对模拟临床血液感染细菌标本的检测，评价该方法的特异性和灵敏度。结果 7个模拟临床血液感染肺炎链球菌标本经AFLP法检测，结果均为阳性；15个模拟临床血液感染非肺炎链球菌标本经该法检测，结果均为阴性；该法检测血液标本中肺炎链球菌的灵敏度为1.5×104cfu/mL。结论 建立的AFLP方法检测血液中肺炎链球菌简便快速，特异性好、灵敏度高，适合肺炎链球菌血流感染的早期诊断。
[关键词] 肺炎链球菌；扩增片段长度多态性；快速检测
[中图分类号] R446.5 [文献标识码] B [文章编号] 1673-9701(2011)34-83-03
Establishment and Application of AFLP Method for Rapid Diagnosis of Streptococcus Pneumoniae in Blood1
YU Lingling1 WANG Xinlin2 WU Xiuji1 ZHENG Xueyun2
1.The Second Affiliated Hospital of Wenzhou Medical College，Wenzhou 325027, China；2.The First People's Hospital of Longwan, Wenzhou 325024, China
[Abstract] Objective To establish a Amplified Fragment Length Polymorphism (AFLP) method for rapid diagnosis of Streptococcus pneumonia. Methods Based on the 16S rRNA nucleic sequence of streptococcus pneumonia, a pair of primers was designed for AFLP. The reaction conditions were optimized, and the specificity, sensitivity, and practicability of AFLP were tested using 7 streptococcus pneumonia strains and 15 non-streptococcus pneumonia strains in blood. Results All the 7 strains of streptococcus pneumonia in blood yielded positive results in AFLP, and the 15 strains of other bacteria in blood all showed negative results. The results of streptococcus pneumonia count showed that AFLP was capable of detecting streptococcus pneumonia at a level as low as 1.5×104cfu/ml in blood. Conclusion The established AFLP method has high specificity and sensitivity for detecting streptococcus pneumonia in blood and is applicable in field of rapid diagnosis of Streptococcus pneumoniae in blood.
[Key words] Streptococcus pneumoniae; Amplified fragment length polymorphism; Rapid detection
肺炎链球菌是一种条件致病菌，当机体免疫力下降时可以致病，肺炎链球菌是社区获得性肺炎、脑膜炎、中耳炎及败血症等的主要病原体[1-2]。血液中检出肺炎链球菌也很常见，国内多见报道。目前对肺炎链球菌的诊断主要依赖传统培养法进行检测，但是培养法需要对微生物分离、培养和鉴定等，检测存在着耗时长、灵敏度低、操作繁琐等缺点，对临床快速诊断尤其是侵袭性感染的诊断不及时，造成临床治疗上的延误。随着分子生物学技术的发展，肺炎链球菌的快速诊断研究进展迅速，有环介导等温核酸扩增技术(LAMP)、PCR检测核糖体(16S rRNA)、荧光定量PCR、荧光探针等[3-6]从基因/基因组的层面对肺炎链球菌进行快速诊断。本研究拟建立扩增片段长度多态性(AFLP)方法以检测肺炎链球菌 ......
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