Preparation Of Visual Biochip For Simultaneous Detection Of Human Immunodeficiency Virus And Treponema Pallidum

Human Immunodeficiency Virus type 1 (HIV-1) and Treponema pallidum (TP) are two kinds of important pathologic organisms for sexual transmitted diseases. They can be transmitted by sexual contact, transfusion and by vertical fetal transmission. The incidence of Acquired Immure Deficiency Syndrome (AIDS) and syphilis is very high. It is difficult to cure these diseases that can do great harm to human. Therefore, it is an important measure to prevent and cure the sexual transmitted diseases and decrease the incidence by diagnosing efficaciously as soon as possible.Many factors such as high cost for preparation and detection system restrict the practice application of the biochip. In recent years, Mirkin, from America northwest university, developed colorimetry analysis method by using nanogold labeling nucleic acid. The detection signal can be amplified 100 million by the combination of immunogold probe and immunogold silver staining enhancement technique. The detection sensitivity can exceed 100 times over fluorescence detection sensitivity. It is speediness and simple, no need for special instruments, and the result can be seen by bare eyes. We purposed to prepare the visual biochip for rapid, nicety, cheap, simple detection for HIV-1 and TP. We have probed into the preparation of visual simultaneous detection gene-chip for HIV-1 and TP, What's more, we also have probed into the preparation of visual protein chip for detection of Tpp47. The work include:1. The preparation of visual simultaneous detection gene-chip for HIV-1 and TPWe chose HIV gag gene and TP Tpp47 gene as target gene according to GeneBank and the relative literatures and have designed probes aimed at target gene. We have examined four controlled experimental factors: the concentration of primer pairs, dNTPs,Mg++ and TaqDNA polymeras, and got preferably results for synchronization amplification for HIV-1 and TP. High conservative amido-modified probes for HTV-1 and TP each were arrayed on aldehyded slides. After the hybridization of biotin-target gene segments and special probes for HTV-1 and TP, the results can be visualized through the exclusively recognition of strepatividin and biotin and gold silver staining enhancement. In order to get more optimized preparation condition, we have studied the specificity and the affect factors on the detection result including probes solvent, hybrdization time, and silver development time.2. The preparation of visual protein chip for detection of Tpp47 We made some alterations during preparation the chip substrate on the basis of the research on treatment with glass surface in our laboratory. After recombinant Treponema pallidum antigen Tpp47 was arrayed on aldehyded slides, specific antibodies in serum were bound to Tpp47, The staphylococcus protein A—modified gold nanoparticle probe exclusively recognized the anti-Tpp47 IgG The "sandwich" format hybridization signal was then amplified with silver staining. We have probed into the capability of nanogold and nanogold probe, the relationship between the immobilizing protein concentration and the gray value, the specificity and the sensitivity. It took 3.5 h to prepare the detection protein chip but only 30 min for detection. The lowest titer of detectable antibody for this method was 1:128, which correlates to about 10.9 U g/ml. Furthermore, the results can be seen unaided, which drastically reduces the cost of detection.In summary, we developed the method for preparation of visual detection biochip, which explored new approach for simple, nicety, cheap detection for sexual transmitted diseases and other clinic pathologic organisms. Also, it has laid the foundation for the research of visual biochip on basic work and clinic applications.