ABSTRACT
Objective To develop a gene microarray system for detection of clinical common pathogenic fungi.MethodsThere were 8 clinical common fungi chosen as the subjects including Candida albicans,Candida glabrata,Candida tropicalis,Candida parapsilokis,Candida pseudotropicalis,Aspergillus terreus,Aspergillus flavus,Aspergillus oryzae,Aspergillus fumigatus.Universal primers,probes and specific probes for the PCR amplification and microarray preparation were designed in ITS region of the fungi genomic DNA.The PCR products amplified from those fungi's genome DNA were denatured and hybridized with the probes in gene microarray.The rapid detection of fungi was based on the investigation on the fluorescent signal intensity in the chip.The detection results of gene microarray system were verified by true positive and negative clinical samples.Results There were totally 25 positive samples identified by clinical routine microbiological methods.The 10 samples identified as bacteria positive were determined as negative without fluorescent signal by the fungi gene microarray,while the 12 samples identified as fungi positive were determined as positive with certain fungus by the fungi gene microarray.And 3 artificial Candida krusei samples were detected as fungi positive,while they were failure to be identified as certain fungus.There was no fluorescent signal in positions of the 8 fungi specific probes,but there was fluorescent signal in the position of fungi universal probe.It indicated that there were fungi in the samples but it couldn't identify the species of the fungi,because the Candida krusei wasn't included in the detection fungi list of the fungi gene microarray.ConclusionsThe fungi gene microarray established by the study could detect the common fungi in clinic rapidly and accurately.This study lays technology foundation for clinical application of gene chip.
ABSTRACT
Objective To detect eight kinds of clinical common pathogenic bacteria by DNA microarray.Methods Eight kinds of common pathogenic bacteria,including Staphylococcus aureus,Pseudomonas aeruginosa,Klebsiella pneumoniae,Escherichia coli,Proteus mirabilis,Enterobacter aerogenes,Pseudomonas fluorescens,Shigella sonnei were collected.Universal primers were designed to amplify 16S rRNA gene fragment from the genomic DNA of the eight bacteria,and probes were designed in the highly variable regions.DNA microarray detection system was established and used for detection of colleted bacteria.A total of 50 samples were collected from the Zhongnan Hospital of Wuhan University,including 6 blood samples,32 sputum samples,9 feces samples and 3 bronchoscope lavage samples.DNA were extracted and detected by the established DNA microarray system.Results The desired fragments were well amplified by the self-designed universal primers.The selected probes had good detection results according to repeated detection.Of the 50 samples detected,pathgenic bacteria were accurately detected in 47 samples.Other three samples were not detected as those bacteria were not included in the chip.By optimizing the detection process,the results could be reported within 8 hours.Observation of probe signal attenuation indicated that even attenuated after 60 days,but the attenuation did not affect the results.Conclusion A microarray system was established for detection of clinical common bacteria accurately and quickly,which provided foundation for its clinical application.