检索历史 清除

目的 确定引起人类感染性腹泻的11种病原微生物,并制备芯片用于检测门诊腹泻患者人粪便标本中的致病菌.方法 根据本院2009年1月至2012年12月期间腹泻门诊的粪便病原菌检测数据,采用生物信息学的方法,收集11种病原菌的所有基因序列,设计引物及探针,优化并制备芯片,PCR扩增杂交并对杂交结果进行分析.用该芯片对本院保存的163个肠道致病菌临床分离株进行鉴定来评价芯片的特异性,用芯片来检测掺有不同浓度沙门氏菌的粪便标本评价芯片的灵敏度.同时收集2010年6月至2013年3月在本院就诊的1 052份腹泻患者粪便标本,平行进行PCR扩增、细菌培养、基因芯片检测,比较不同检测方法的阳性率.结果 成功制备了腹泻相关11种致病菌检测芯片.应用制备的芯片检测了163个临床分离株,准确率达100％.与PCR方法比较,基因芯片检测沙门氏菌的灵敏度达102 CFU/ml,比PCR法检测灵敏度高10倍.用该芯片对临床1 052份腹泻患者腹泻标本进行检测,与传统的培养法及PCR法比较,有较高的阳性检出率,达36％,比常规细菌培养阳性率高13％(x2=2.28,P＜0.05),比PCR检出率高4％(x2=5.16,P＞0.05).结论 成功制备11种腹泻相关致病菌基因芯片,能同时对11种腹泻致病菌进行检测,有较好的特异性和灵敏度,有更高的阳性率,可以用于临床检测.

Objective To determine 11 kinds of pathogenic microorganisms related with human infectious diarrhea,and prepare biochips to detect pathogenic bacteria in stool samples of diarrhea patients in outpatient clinic.Methods According to the data of pathogenic bacteria detection in stool samples in diarrhea clinics of our hospital between Jan uary 2009 to December 2012,bioinformatic method was used for collecting whole sequences of 11 species of pathogenic bacteria,designing primers and probes,optimizing and preparing chips.PCR experiments were performed and amplification results were analyszd.The specificity of these chips was evaluated by detecting 163 strains of intestinal bacteria isolated from diarrhea patients,and sensitivity of chips was accessed by detecting stool samples mixed with different concentrations of Salmonella.1 052 stool samples were collected from diarrhea patients from June 2010 to March 2013 in our hospital.PCR,bacterial culture and DNA microarray chip were performed on these samples in order to compare the positive rates of different detection methods.Results Detection chips of 11 kinds of diarrheaassociated pathogens were successfully prepared.The accuracy rate 163 of clinical isolated strains detected by these chips was 100％.When Salmonella was detected,sensitivity of chips was 102 CFU/ml,which was 10 times greater than the PCR assay.The positive tate of 1 052 clinical diarrheal samples was tested by these chips was 36％,better than that of the traditional bacteria culture and PCR assay,which was 13％ higher than that of traditional bacteria culture (x2=2.28,P＜0.05) and 4％ higher thanthat of PCR assay (x2=5.16,P＞ 0.05).Conclusions Microarray chips of 11 kinds of diarrhea-associated pathogens is successfully prepared,which can detect 11 kinds of pathogenic bacteria of diarrhea simultaneously,with good specificity and sensitivity,and higher positive.This method can be used in clinical practice.