焦磷酸测序技术快速鉴定体液病原菌方法的建立与应用

• 作 者: 任绪义 林洁 虞闰六 章静 吕江峰
• 机 构: 310030,杭州迪安医学检验中心,浙江大学医学院附属邵逸夫医院检验科,310030,杭州迪安医学检验中心,310030,杭州迪安医学检验中心,310030,杭州迪安医学检验中心
• 发表日期: 2011年4期
• 关键词: 体液 病原 生物学鉴定法 序列分析

目的 建立一套快速准确鉴定临床体液样本病原菌的方法学体系,评价其作为病原菌检测方法在临床检验中的应用价值.方法 采用柱式抽提试剂盒抽提205份体液样本中病原菌基因组dna,以细菌16s rrna基因为目标序列,通用引物扩增高变区片段,以焦磷酸测序技术进行扩增子序列分析并与数据库中的序列进行比对和病原菌种属鉴定.同时采用传统的细菌培养-生化方法进行病原菌检测鉴定,并以此为金标准,采用spss 11.0软件计算焦磷酸测序法检测敏感度、特异度、假阳性率、假阴性率、阳性预测值、阴性预测值、阳性似然比及阴性似然比,评价新方法的可行性与实用性.结果 细菌培养阳性率为39.5％( 81/205),其中71例为单一病原菌感染,10例为双重混合感染；与培养-生化鉴定结果相比,焦磷酸测序检测鉴定单一病原菌感染样本结果符合率为100％(71/71),鉴定双重混合感染样本10例中有7例符合.此外,对培养阴性的124例体液样本用焦磷酸测序检测鉴定,结果10例为阳性,且均为单一细菌感染.以培养方法作为金标准,焦磷酸测序法鉴定体液病原菌敏感度达100％,特异度为91.9％,假阳性率为8.1％,假阴性率为0％,阳性预测值为89.0％,阴性预测值为100％,阳性似然比为12.4,阴性似然比为0.结论 焦磷酸测序法直接检测鉴定临床体液样本中的病原菌具有快速、高敏感性、高准确率等优势,在临床病原微生物检测中具有很强的实用性和广阔的应用前景.; abstract：; objective to develop a method for rapid and accurate detection and identification of bacterial pathogens directly from body fluid specimens and to evaluate its application in clinical laboratory.methods bacteria dna was extracted from 205 body fluid specimens with column-based kit,and the high variable v1 and v3 regions of bacterial 16s rrna gene were amplified with broad-range primers.amplicons were analyzed by pyrosequencing and the generated sequences were searched in the bacterial identification database.traditional culture-biochemical method was also used for these specimens and the results were taken as the golden standard.spss 11.0 was used to calculate the sensitivity,specificity,false positive/negative rate,positive/negative predictive value and positive/negative likelihood rate of pyrosequencing method.results the positive rate of bacteria culture was 39.5％ (81/205),among which 71 were infected with single bacterium,and 10 were infected with two species of bacteria.compared with the culture identification results,pyrosequencing had a 100.0％ (71/71) concordance when applied to detect and identify bacterial pathogens from specimens with single specie bacterium infected.to specimens with two species bacteria infected,7 out of 10 specimens were in concordance with the culture identification results.besides,pyrosequencing detected 10 positive specimens and identified bacterial pathogens infected in the 124 culture-negative specimens.taken bacteria culture as the standard method,the sensitivity of pyrosequencing for identifying bacterial pathogen in body fluid was 100.0％,and with a specificity of 91.9％,the false positive rate was 8.1％,the false negative rate was 0.0％,the positive predictive value was 89.0％,the negative predictive value was 100.0％,and the positive and the negative likelihood rate were 12.4 and 0,respectively.conclusion pyrosequencing can be used to detect and identify bacterial pathogens directly from body fluid specimens with the advantages of rapidity,high sensitivity,high accuracy and high throughput.