| 巢式聚合酶链式反应检测脑脊液标本中病原体的方法评价 |
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| 引用本文： | 田国忠. 巢式聚合酶链式反应检测脑脊液标本中病原体的方法评价[J]. 疾病监测, 2020, 35(2): 146-150. DOI: 10.3784/j.issn.1003-9961.2020.02.014 |
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| 作者姓名： | 田国忠 |
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| 作者单位： | 中国疾病预防控制中心传染病预防控制所，北京 102206 |
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| 摘 要： | 目的探讨巢式聚合酶链式反应(PCR)检测脑脊液标本中病原体的可行性,为疾病的快速临床诊断提供参考。方法源自细菌16S rRNA基因序列设计的巢式PCR技术方法,包括2对引物,2次PCR扩增,第1对引物首次PCR扩增脑脊液标本提取的核酸DNA,第2对引物再次PCR扩增,其DNA模板为第1轮PCR扩增产物。将第2轮PCR扩增产物进行测序,对序列进行比对分析,从而确定感染的病原体。使用DNA微量分光光度测定布鲁氏菌纯菌核酸DNA,将DNA进行倍比稀释,用于巢式PCR敏感性测试。结果巢式PCR能够检测的最低限约为1个核酸DNA拷贝数。应用巢式PCR检测40份临床患者的脑脊液标本,扩增结果表明有37份标本获得约1 460 bp的预期扩增条带(不同细菌扩增片段有差异),测序比对结果显示,检出脑膜炎奈瑟菌7份、产碱假单胞菌1份、草假单胞菌22份、嗜麦芽窄食单胞菌2份、肺炎链球菌1份、未知细菌性病原体4份、未检出3份。结论巢式PCR能够快速检测与鉴定脑脊液标本中的细菌性病原体。
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| 关 键 词： | 巢式聚合酶链式反应 16S rRNA 脑脊液 细菌性病原体 |
| 收稿时间： | 2019-11-03 |
Evaluation of nested polymerase chain reaction in detection of pathogens in cerebrospinal fluid |
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| Tian Guozhong. Evaluation of nested polymerase chain reaction in detection of pathogens in cerebrospinal fluid[J]. Disease Surveillance, 2020, 35(2): 146-150. DOI: 10.3784/j.issn.1003-9961.2020.02.014 |
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| Authors: | Tian Guozhong |
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| Affiliation: | National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China |
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| Abstract: | ObjectiveTo evaluate the feasibility of detecting pathogens in cerebrospinal fluid by nested polymerase chain reaction (PCR), and provide the reference for rapid and accurate diagnosis of diseases in clinical practice.MethodsA nested-PCR was designed by using the bacterial 16S rRNA gene sequences, including two PCR amplification processes by using two pairs primers. The bacterial DNA, which was extracted from cerebrospinal fluid, was amplified firstly by PCR using first pair primers. And the second PCR amplification was followed using the second pair primers. The DNA template was the first PCR amplification products. The products of the second PCR amplification were sequenced. The pathogen was determined by comparing and analyzing the sequences. The concentration of pure Brucella DNA was determined by DNA spectrophotometry. The sensitivity of nested-PCR was evaluated by using serial dilutions of DNA template. Two-fold serial dilutions were prepared from bacterial suspension.ResultsThe minimum detection limit of nested-PCR was about one copy number of DNA. The cerebrospinal fluid samples of clinical patients were detected by using nested-PCR. In 40 clinical cerebrospinal fluid samples, about 1 460 bp electrophoresis strips were detected in 37 samples. The sequencing results indicated that 7 cases were Neisseria meningitidis, 1 case was Pseudomonas alcaligenes infections, 22 cases were Pseudomonas poae infections. 2 cases were Stenotrophomonas maltophilia infections. 1 case was Streptococcus pneumoniae infection. 4 cases were unknown bacterial infections, and 3 cases had negative bacterial DNA detection results.ConclusionThe nested-PCR can be used in rapid detection of bacterial pathogens in cerebrospinal fluid with accurate results. |
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| Keywords: | Nested-polymerase chain reaction 16S rRNA Cerebrospinal fluid Bacterial pathogen |
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