Sexually Transmitted Disease Pathogen Detection, Gene Chip Study

In this paper, an oligonucleotide microarray based on the multiplex asymmetric PCR with Cy3 labelled at the 5'-end of each reverse primer was made, which makes it possible to detect and identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) common in clinics of sexually transmitted diseases (STD) in one tube and in a large scale.It was proved from the study on the fabrication of the oligonucleotide microarray that the best way to preparation of the microarray for diagnosis of the pathogens responsible for STD was to spot the 40mer oligonucleotide probes with the bifunctional modification onto the aldehyde slides.The primers and the probes specific for the genes of NG, CT, UU and an internal control (Luciferase, Luc) as well as a probe specific for the lectin gene used as a negative control were designed by the software Primer Primer5.0 and DNASIS 5.0. The specificities of the primers and the probes and the feasibility for the microarray to detect the multiple pathogens responsible for STD in one tube were proved by the uniplex PCR and the multiplex PCR for amplification of the plasmids of NG, CT, UU and Luc. It was indicated from the optimization on preparation of the microarray that the best way to prepare the microarray for detection of the pathogens responsible for STD was to use the spotting solution A to spot the probe which is closed to the end of Cy3-labelled primer in a gene to be amplified onto the aldehyde slides at a concentration of 50uM and mix the hybridization solution B with the PCR products heated at 100C for 10 min at the ratio of 2:1 for hybridization on the slides at 42C for lh.The optimization on the sensitivity of the microarray proved that the best amplification condition for the multiplex PCR in detection of the pathogens responsible for STD was that the forward and reverse primer concentrations of NG, CT, UU and Luc were 0.225uM, 0.135uM, 0.15uM, .3uM and 2.25uM, 1.35uM, 1.5uM, 3uM respectively in a 25ul-reaction system. In the system, the concentrations of dNTP,MgCl2and KC1 were 300 uM,3mM and 75mM and the Taq DNA polymerase was 2U. The PCR amplification was carried out in a 1 xPCR buffer under the following condition: preheating (5 min/94C); 35cycles: denature (30 sec/94C), anealing(30 sec/57C), extension(30 sec/72C); extension (5 min/72C). When the genes of NG, CT, UU and Luc were amplified at this condition, the oligonucleotide microarray got its highest sensitivity of 5x103copies for each gene in detection of NG, CT, UU and Luc plasmids. The evaluation on the performance of the microarray for detection of the pathogens responsible for STD indicated that the oligonucleotide microarray was specific for the pathogens of NG, CT, UU. In diagnosis of NG, CT and UU in 60 samples from STD clinics, the microarray method took a coincidence of 97.2%, 100% and 75.2% in detection of NG, CT and UU, respectively when compared with the PCR used in the clinic practice.