DNA microarray for simultaneous screening and detection of seven rickettsia / 军事医学

ABSTRACT

Objective To develop a chemiluminescence ( CL ) imaging DNA microarray method for simultaneous detection of seven rickettsiae.Methods Primers and probes were designed based on the specific sequence of seven rickettsia genomes.The probes were immobilized on the aldehyde modified glass surface to prepare DNA microarray for rickettsiae.The nucleic acids of the selected rickettsiae were amplified and labelled by multiplex PCR method, and then hybridized with microarray that was scanned after washing and chemiluminescence coloration, before the results were analyzed.Facilitated by the optimization of the multiplex PCR system, hybridization, and chemiluminescence imagination, we evaluated the specificity,sensitivity and reproducibility of the chip.The serial diluted nucleic acid of Rickettsia mooseri was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Double-blind simulated samples were prepared to further evaluate the accuracy of the detection methods.Results One universal primer, four specific primers, one universal probe, and nine specific probes were selected.This DNA microarray demonstrated high specificity and sensitivity.The detection sensitivity of plasmid DNA and double-blind simulated sample DNA was 1.5 ×102 -3 ×103 copies per reaction and 103 -104 copies/μl.The detection results of real-time PCR method was consistent with the microarray, and the microarray possessed 10 fold lower detection sensitivities than the real-time PCR method.The coincidence rate of double-blind simulated sample detection was 100%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous detection of seven rickettsiae is established successfully,which can serve as a new high throuthput detection method for clinical diagnosis and epidemiological investigation of rickettsiae.