Preparation Of Armored RNA Virus-like Particles And The Application Of AIV Detection By Real-time PCR

Armored RNA technology is a new technique of preparation for RNA quality control, which is developed in recent years. The principle is that E.coli bacteriophage MS2 coat protein gene and foreign fragment was cloned into the expression vector by genetic engineering methods, and the foreign fragment can be transcripted into recombinant RNA. The recombinant RNA is packaged by MS2 coat protein as RNA-protein complex. This spherical RNA-protein complex was named as virus-like particles. Now Armored RNA technology is gradually applied to the research of detection of RNA viruses.Firstly, according to the principle of Armored RNA technology, a vector that can express RNase-resistant virus-like particles was constructed. The Foot-and-mouth disease virus IRES RNA fragment was selected as the internal standard gene. The gene was cloned into the expression vector, then the prokaryotic expression vector expressing virus-like particles was obtained and was induced. The expression products was confirmed by the enzyme digestion assay. The virus-like particles RNA was extracted and was identified by RT-PCR. Then we confirmed that the expression products were virus-like particles containing foot-and-mouth disease virus IRES RNA. The expression products were purified by sucrose density gradient centrifugation and were observed by transmission electron microscope. A series of experimental results showed that the expression products were circular virus-like particles containing foot-and-mouth disease virus IRES RNA. The diameter of the virus-like particles is about 26nm. The prepared virus-like particles with good stability are easy to preserve because of the RNase - resistant characteristic.Secondly, the IRES gene was selected as the target, and we tested the virus-like particles RNA by double-stranded fluorescent probes in one-step reverse transcript real-time fluorescence PCR assay. Then we optimized the reaction systems and the reaction conditions. The results showed that double-stranded fluorescent probes could test the virus-like particles IRES RNA in the optimal one-step reverse transcript real-time fluorescence PCR assay. Finally, the prepared virus-like particles was selected as internal control, and avian influenza virus was tested through adjusting the reaction system in real-time fluorescence PCR assay. The experiment results showed that avian influenza virus could be quickly tested by real-time PCR. And because of the internal control, we can also avoid the false negative results caused by inhibitors and human error in the assay or other factors.The virus-like particles containing foot-and-mouth disease virus IRES RNA prepared in the study were tested by a series of experiments and applied to the detection assay of avian influenza virus. From these experiments, we can know that the structure of the virus-like particles is similar as RNA virus. Both of them are RNA-protein complexes with RNA wrapped by coat proteins. So we can put the virus-like particles together with other virus, then they experience RNA extraction, reverse transcription PCR amplification and detection in order to monitor the whole process and avoid false negative results. The virus-like particles with RNase - resistant characteristics have good stability. They are also secure and easy to store. Therefore, The virus-like particles prepared in the study provide a good platform for the detection of RNA viruses.