利用限制性片段长度多态性检测脑脊液中的细菌病原体
Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism.
作者信息
Kalghatgi A T, Praharaj A K, Sahni A K, Pradhan D, Kumaravelu S, Prasad P L, Nagendra A
机构信息
Senior Advisor (Pathology), Command Hospital (Central Command), Lucknow.
PDMS (HS) DGMS Navy, Naval HQ, New Delhi.
出版信息
Med J Armed Forces India. 2008 Jan;64(1):29-32. doi: 10.1016/S0377-1237(08)80141-4. Epub 2011 Jul 21.
BACKGROUND
Polymerase chain reaction (PCR) is useful for rapid microbial detection in body fluids with low microbial load. It is easier to use universal or broad range primers for the amplification of conserved stretches of DNA common to all bacteria like 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) of PCR products.
METHODS
Forty samples of cerebrospinal fluid were collected. After DNA extraction, universal or broad range PCR was performed using two universal primers U1-5'-CCAGCAGCCGCGGTAATACG-3', corresponding to nucleotides 518 to 537 of the Escherichia coli 16S rRNA gene, and U2 - 5'-ATCGG(C/T)TACCTTGTTACGACTTC-3', corresponding to nucleotides 1513 to 1491 of the same gene. The PCR product was subjected to digestion by endonucleases- HaeIII, Mn11, BstB1 and Alu1. Restriction pattern obtained was compared with that of standard organisms to identify the pathogen. The results were compared with conventional methods.
RESULT
Universal PCR could detect pathogens in 20% samples within 13-18 hours as compared to 16% by conventional methods. The analytical sensitivity was 10 Gram negative and 250 Gram positive organisms per 200 μl sample. Overall sensitivity was 83.3% and specificity was 91.2%.
CONCLUSION
Universal PCR followed by RFLP of PCR product is a good alternative to conventional diagnosis of bacterial pathogens.
背景
聚合酶链反应（PCR）有助于在微生物载量低的体液中快速检测微生物。使用通用或广泛引物扩增所有细菌共有的保守DNA片段（如16S rRNA基因），然后对PCR产物进行限制性片段长度多态性分析（RFLP）会更加简便。
方法
收集40份脑脊液样本。提取DNA后，使用两种通用引物进行通用或广泛PCR，引物U1 - 5'-CCAGCAGCCGCGGTAATACG-3'，对应大肠杆菌16S rRNA基因的核苷酸518至537，引物U2 - 5'-ATCGG(C/T)TACCTTGTTACGACTTC-3'，对应同一基因的核苷酸1513至1491。PCR产物用核酸内切酶HaeIII、Mn11、BstB1和Alu1进行消化。将获得的限制性图谱与标准生物体的图谱进行比较以鉴定病原体。将结果与传统方法进行比较。
结果
通用PCR可在13 - 18小时内检测出20%样本中的病原体，而传统方法为16%。分析灵敏度为每200μl样本中有10个革兰氏阴性菌和250个革兰氏阳性菌。总体灵敏度为83.3%，特异性为91.2%。
结论
通用PCR及随后的PCR产物RFLP分析是细菌病原体传统诊断的良好替代方法。
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