16SrDNA测序在儿童自发性细菌性腹膜炎病原学诊断中的作用

摘要

目的：探讨Sanger法测序用于快速鉴定儿童自发性细菌性腹膜炎病原菌的价值。方法用临床常见的16种细菌和3种真菌及人类基因组DNA验证一对新的16S rDNA通用引物PSL/P13P的特异性和敏感性；收集86例疑似自发性细菌性腹膜炎儿童的腹水标本5 ml,其中4 ml常规细菌培养,剩余1 ml用于提取细菌DNA进行16S rDNA-PCR；对阳性扩增产物进行测序并通过BLAST比对鉴定菌种,与培养鉴定结果进行比较。结果3种真菌及人类基因组PCR均为阴性,16种细菌PCR阳性,PSL/P13P的检测下限为102 CFU/ml。86例标本培养阳性率26.7%(23/86),PCR阳性率45.3%(39/86),二者差异具有统计学意义(χ2=14.06,P<0.05)。在37例PCR及测序阳性标本中,革兰阴性杆菌占73.0%(27/37),以大肠埃希菌为主(14/37,占37.8%),革兰阳性球菌占27.0%(10/37),其中肠球菌最多(7/37,占18.9%)。结论通用引物PSL/P13P具有良好的特异性和敏感性,检出率较常规培养方法高,结合测序能很好地鉴定自发性细菌性腹膜炎病原菌,可作为一种新的检测技术和流行病学方法在临床应用；儿童自发性细菌性腹膜炎病原菌以革兰阴性杆菌为主,尤以大肠埃希菌最常见。%Objective To explore the value of Sanger sequencing for rapidly identifying pathogenic bacteria-caused spontaneous bacte-rial peritonitis(SBP) in children. Methods The specificity and sensitivity of a new pair of 16S rDNA primer(PSL/P13P) was vali-dated using 16 kinds of bacteria, 3 kinds of fungi and human DNA. Eighty-six children with suspected SBP were included. The 5 ml ascetic fluid was collected, of which, 4 ml ascetic fluid was cultured and the rest 1 ml was used for DNA extraction following by 16S rDNA-PCR. The positive PCR results were sequenced and identified by BLAST, and then compared with the culture results. Results PCR products from 3 kinds of fungi and human DNA were negative, and PCR products from 16 kinds of bacteria were positive. The lowest detectable limit of PSL/P13P was 102 CFU/ml. Of 86 cases of ascites, the positive rate was 26. 7%(23/86) for culture results and 45. 3%(39/86) for PCR results, and there was a significant difference between the two methods(χ2 =14. 06,P<0. 05). In 37 samples of positive PCR and sequencing results, Gram-negative bacillus accounted for 73. 0%(27/37), which mainly was Escherichia coli(14/37,37. 8%);Gram-positive coccus accounted for 27. 0%, and the predominant pathogen was Enterococcus(7/37,18. 9%). Conclusion The universal primers PSL/P13P has good specificity, sensitivity and higher detection rate, and its combination with se-quencing can detect pathogenic bacteria-caused SBP very well. So it can be used as a new detection technology and epidemiological in-vestigation method in the clinical practice. The pathogens causing SBP in children are mainly Gram-negative bacteria, and Escherichia coli is the most common one.