Multiplex Bead Array Assays Applied To The Diagnosis Research Of Viral Disease

In recent years, Luminex Corporation has developed a high-throughput detection technology, which called Multiplex Bead Array Assays. This technology puts fluorescent coding microspheres as carrier, integrates technologies such as laser analysis, fluidics, high speed digital signal processing and computer algorithms. Its biggest characteristic is to realize rapid detection of 100 kinds of indicators at the same time, and its target detection object covers a variety of biological molecules, such as antibody, nucleic acid and cell factor. Multiplex Bead Array Assays was considered as one of the trend clinical diagnosis technologies.The objective of this study was applying Multiplex Bead Array Assays to the diagnosis research of viral disease. In the first part of this research, we through the application to establish a rapid, sensitive and high-throughput laboratory testing method of viral hemorrhagic fever, in order to realize rapid screening or diagnosis of common viral hemorrhagic fever in China. At the first, we coupled 13 kinds of purified recombinant protein of hemorrhagic fever virus to 13 fluorescent microspheres, respectively, and through the detection with corresponding polyclonal rabbit serum to evaluate protein coupling effect. Then we used these 13 protein-coupled fluorescent microspheres to detect Ig M and Ig G of 96 normal serums to determine the cutoff value of the multiple serological detection method. To evaluate effect in common viral hemorrhagic fever disease detection of this method, we detected 71 HFRS acute phase serum and 73 HFRS convalescent serum, 41 SFTS acute phase serum and 41 SFTS convalescent serum, 50 DENV acute phase serum and 64 DENV convalescent serum, all serum above were confirmed as positive serum by RT-PCR or ELISA. The test results showed that our established 13 microspheres chip detection method had high sensitivity and specificity, whether in acute phase serum detection or convalescent serum detection, the coincidence rate was high, and the Kappa value was greater than or equal to 0.75, and the degree of consistency is ideal.The success of applying Multiplex Bead Array Assays to detect common hemorrhagic fever in China let us believe that establish a rapid, sensitive and high-throughput laboratory testing method of viral hemorrhagic fever may be possible. At the same time, we also try to apply this technology to another kind of viral diseases detection work, the HFMD, which caused by enterovirus. EV71 is one of the important pathogens of HFMD. As in our study of Multiplex Bead Array Assays application, the expression and purification of viral protein is pivotal, so in the second part of this research, we used prokaryotic expression system to express EV71 VP1 recombinant protein. After the protein purification, we also identified the antigen activity of VP1 recombinant protein.To express the EV71 VP1 recombinant protein, we extracted the viral RNA, and amplified the full-length VP1 gene by using RT-PCR method. The RT-PCR product was inserted into p MD20-T vector, and confirmed by sequencing. The complete encoding VP1 region was amplified by primer pairs designed for its expression in vitro, and then cloned into p ET32a(+) vector. The recombinant plasmid was then transformed into E. coli BL21(DE3). After induction by using IPTG, the bacterial lysate were electrophoresis by SDS-PAGE, and the antigen activity was identified by Western Blotting. The result showed that the VP1 recombinant protein was expressed successfully, and the molecular weight was in line with expectation. The Western blotting test showed the VP1 recombinant protein possessed strong antigen activity.At this point, we had set up a complete protein expression system in our laboratory, prepared the technology for the following other enterovirus protein expression, and provided the conditions of applying Multiplex Bead Array Assays to establish a rapid, sensitive and high-throughput laboratory testing method of HFMD or any other viral diseases. In fact, the VP1 recombinant protein can be study further to apply to serum epidemiology survey of EV71 infection, or to apply to monoclonal antibody research. The success of VP1 recombinant protein expression also laid the important foundation for developing EV71 vaccine or developing rapid diagnosis reagents at early stage of virus infection.