Phage Display Mediated Amplification Methods For Pathogen Detection

The detection of pathogenic microbes is a great challenge because of the severesituation of infectious diseases and potential threaten of bioterrorism. A new detectionmethod for pathogen, called phage display mediated amplification method, wasdeveloped in this study. This method takes advantage of the recombinant phage,which displays antibody on thephage surface and includes DNA inside the phage.The surface displayed single chain variable fragment (scFv)and DNA inside thephage themselves can directly serve as detection antibody and nucleic acidamplification template, respectively. Thus, the recombinant phage, as a vector,mediates the process of immunological recognization and signal detection. Two kindsof methods, phage display mediated immuno-PCR and phage display mediatedisothermal amplification, were developed in this study based on the difference ofamplification method.Phage display mediated immuno-PCR (PD-IPCR)was a newtype of immuno-PCR method, in which the recombinant phage is a novel vector forimmuno-PCR. Hantaan virus nucleocapsid protein (NP) was selected as a model. Theresults demonstrated that the PD-IPCR method was high sensitive with a detectionlimit of 10 pg/ml for NP, which corresponded to an about 1,000-10,000-fold increasein sensitivity as compared to the enzyme-linked immunosorbent assays (ELISA)under analogous conditions. The real-time PCR technology was also applied in thismethod, which enabled the quantitative detection for antigen. The linear regressionwas good (R~2=0.96)for the signals of spiked samples against the logarithmicconcentrations from 2,000 ng/ml to 2 ng/ml in real-time sandwich PD-IPCR.Isothermal amplification methods were applied in the further study to develop phagedisplay mediated isothermal amplification methods for more broad applicationswithout relying on complicated machines. Rolling circle amplification (RCA) wasapplied first. The sensitivity of phage display mediated immuno-RCA for HIV P24antigen was 100 pg/ml. However, the amplification time of RCA is long and thespecificity is not high as other amplification methods. So, strand displacementamplification (SDA) was applied as the signal amplification to develop phage displaymediated immuno-SDA. The amplification efficiency of SDA reaction is so high thatthe reaction time is only 1h. The detection sensitivity for HIV P24 antigen and HBVHBsAg was increased 1,000 to 10,000 folds compared with conventional ELISA.FRET fluorescent probe was applied to develop real-time assay, which increase theassay specificity largely. The phage display mediated amplification methodsdeveloped in this study share main advantages of phage display and nucleic acid amplification technology. With the high sensitivity, specificity and low cost, thesemethods will be a routine technique in pathogen detection.