通用基因芯片检测感染性细菌方法的研究

目的：为提高临床微生物的检测速度和准确性，建立一次实验就能检测出一个样品中多个目标的方法。方法：利用生物信息学手段设计16种常见临床细菌的寡核苷酸探针，对16种临床常见微生物进行检测。结果：建立以16S rDNA为对象的基因芯片检测方法。利用寡核苷酸探针芯片对16种临床常见细菌进行检测的结果证明，大部分的细菌都可以由4条探针确定到种的位置。纯细菌菌液最低可以检测到200（铜绿假单胞菌）到235（金黄色葡萄球菌）个细胞。血液模拟标本只能得到2000到23500个细胞。染色体系列稀释灵敏度实验表明基因芯片检测比PCR－电泳灵敏10倍。混合标本盲测实验结果表明，样品中如果存在一个以上的待测对象也可以检测出来。结论：利用寡核苷酸为探针的基本芯片检测系统具有一定的种属鉴别能力，可以将本实验中提及的大部分细菌区分到种的位置。整个检测过程大约需要4．5h。

Study on universal microarray system for detection of common clinical infectious bacteria

Objective For the rapid and efficient detection of biological agents and clinical infectious bacteria,a based detection system which employed 16SrDNA sequence as its detection target was etablished.Methods Oligonucleotide probes of 16 strains of bacteria were designed by using some bioinformatics softwares and methods and tested by using this microarray system.Results A microarray based detection system was successfully developed.The results showed that most of the bacteria can be determined at the species level.The detection limit for oligonucleotide microarray system was 200 CFU for Pseudomonas aeruginosa and 235 CFU for Staphylococcus aureus. Aliquots of bacterial suspension were spiked into horse blood,and subject to analyse with microarray system,the sensitivity was greatly decreased to 2 000 CFU of Pseudomonas aeruginosa and 23 500 CFU of Staphylococcus aureus. But with the serial dilution of Legionella pneumoniae chromosomal DNA,a 10 times sensitivity than that of PCR-electrophoresis was observed.As for the bland test for pooled samples,two strains of bacteria in a single sample could be deteced by this method at the same time.Conclusion The oligonucleotide microarray could determine most of the test strains at species level.The overall time for sample process,hybridization and data acquisition lasted about 4.5 hours.