穿刺引流液中病原性真菌的ITS测序研究

Detection of the pathogenic fungi from the puncture fluid by sequencing the ITS region

目的探讨内部转录间隔区(ITS)测序用于快速检测穿刺引流液中病原性真菌的价值。方法采用临床常见的14种细菌和4种真菌及人类基因组DNA验证真菌通用引物(ITS1/ITS4和ITS3/ITS4)的特异性和敏感性;采集90例临床疑似真菌感染患者的穿刺引流液标本10mL,其中8mL用于常规真菌培养,剩余2mL用于提取真菌DNA并分别用ITS1/ITS4和ITS3/ITS4作为引物进行扩增,将阳性扩增产物测序并与BLAST比对,将测序法与培养法的阳性率结果进行统计学比较。结果14种细菌及人类基因组扩增产物均为阴性,4种真菌扩增产物为阳性。ITS3/ITS4和ITS1/ITS4的最低检测限分别为102CFU/mL和103 CFU/mL。90例标本培养阳性率为4.44%(4/90),测序法阳性率为12.22%(11/90),二者差异具有统计学意义(P<0.05)。结论引物ITS1/ITS4和ITS3/ITS4的特异性良好,后者较前者扩增敏感性更高;测序真菌ITS区可作为临床快速检测穿刺引流液中病原性真菌感染的方法。

Objective To explore the value of sequencing internal transcribed spacer（ITS）in identification of pathogenic fungi species from the puncture fluid.Methods The specificities and sensitivities of primers（ITS1/ITS4 and ITS3/ITS4）were validated by using 14 kinds of bacteria,4 kinds of fungi and human DNA.90 cases of clinical patients with suspected fungal infections were enrolled.10 mL puncture drainage fluid was collected from each patient,and 8 mL in which was cultured and the rest 2 mL was used for DNA extraction and PCR detection with ITS1/ITS4 and ITS3/ITS4 as primers.The positive PCR products were compared with BLAST,and the results were analyzed statistically.Results PCR products of bacteria and human DNA were negative,which of fungi were positive.The lowest detectable limits of ITS1/ITS4 and ITS3/ITS4 were 103 CFU/mL and 102 CFU/mL,respective-ly.Of the 90 cases of puncture drainage fluid samples,the cultivation positive rate was 4.44%（4/90）,PCR positive rate was 12.22%（11/90）,and the difference was statistically significant（P〈0.05）.Conclusion ITS1/ITS4 and ITS3/ITS4 are well in spe-cificity,but sensitivity of ITS3/ITS4 is higher than ITS1/ITS4 .ITS region sequencing will become a quick method of detecting fun-gal infection in the puncture fluid.