CAJ | 학술논문

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.
목적 건립일투쾌속준학감정림상체액양본병원균적방법학체계,평개기작위병원균검측방법재림상검험중적응용개치.방법 채용주식추제시제합추제205빈체액양본중병원균기인조DNA,이세균16S rRNA기인위목표서렬,통용인물확증고변구편단,이초린산측서기술진행확증자서렬분석병여수거고중적서렬진행비대화병원균충속감정.동시채용전통적세균배양-생화방법진행병원균검측감정,병이차위금표준,채용SPSS 11.0연건계산초린산측서법검측민감도、특이도、가양성솔、가음성솔、양성예측치、음성예측치、양성사연비급음성사연비,평개신방법적가행성여실용성.결과 세균배양양성솔위39.5％( 81/205),기중71례위단일병원균감염,10례위쌍중혼합감염；여배양-생화감정결과상비,초린산측서검측감정단일병원균감염양본결과부합솔위100％(71/71),감정쌍중혼합감염양본10례중유7례부합.차외,대배양음성적124례체액양본용초린산측서검측감정,결과10례위양성,차균위단일세균감염.이배양방법작위금표준,초린산측서법감정체액병원균민감도체100％,특이도위91.9％,가양성솔위8.1％,가음성솔위0％,양성예측치위89.0％,음성예측치위100％,양성사연비위12.4,음성사연비위0.결론 초린산측서법직접검측감정림상체액양본중적병원균구유쾌속、고민감성、고준학솔등우세,재림상병원미생물검측중구유흔강적실용성화엄활적응용전경.
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.