The Study Of Multiplex RT-PCR-based Reverse Dot Blot Hybridization Technique For Detecting Influenza Viruses

Influenza, a acute respiratory disease, which is caused by A, B, C influenza viruses. Influenza viruses can cause much more serious diseases than the common cold.It is easy to be infected and can cause a variety of complications. It may even lead to world pandemic.The harm to human, bird and beast has drawn increasing attention in general. Nowadays, the mainly methods for detecting influenza viruses in laboratory are virus isolation, serological testing and DNA testing. But there are some limitations, using chick embryo to isolate influenza viruses spends more time and always requires high-level laboratories. Serological test can not get the result immediately. Because existed nucleic acid testing can only identify one specific type of influenza viruses at once, it is Complex, time-consuming and costly.A method, multiplex RT-PCR-based reverse dot blot hybridization technique, was established for detecting influenza viruses. This method can rapidly detect multiple influenza viruses at the same time in one reaction. To establish this method need to do the following three aspects.1. To establish the multiplex RT-PCR System. The target genes of influenza virus were determined. The primers and probes for influenza virus detection were designed. All primers have a 5'NH2 (C12) tag and all probes have a 5'BIOTIN tag. The concentration of primers was explored, the best concentration for this study is 0.2μM .And the designed probes and primers were selected. The specificity of established multiple RT-PCR system was detected, it has high specificity.2. To set up reverse dot hybridization system. T plasmid which conclude full-length target gene was constructed. The nucleic acid low-density chips for detecting influenza viruses were prepared. The specificity of this system was detected.. It can successfully detect and identify influenza viruses. This assay is 100-1000 times more sensitive than traditionally RT-PCR method.3. The established method was assessed by clinical samples. Use the established method to detect 50 clinical samples which were collected from a suspected influenza outbroken middle school, to real-time RT-PCR test results from National Influenza Centre as the standard, Sensitivity was 95%, specificity of 100%.This established method can clearly reed the result with visible naked eyes, and the background is clean.It is very objective and accurate. The key to this study is the specific primers and probes. The probe of influenza A was designed through analysis the M gene, the probe of influenza B was designed through analysis the NS gene.Then the specific probes of different subtypes influenza A viruses was designed through analysis the HA gene. After this, the accuracy of this established method was increased.The results show that this established method can effectively detect influenza viruses. It can detect multiple subtypes of influenza viruses at one time. It is useful to rapidly diagnosis the influenza virus. It also can be used to monitor the influenza. This study has successfully established a method, multiplex RT-PCR-based reverse dot blot hybridization technique, for detecting influenza viruses.