The Study On The Rapid Detection Of Avian Influenza Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification

Avian influenza is a respiratory disease in poultry caused by RNA viruses of the family Orthomyxoviridae, the avian influenza viruses (AIV).Since first reported in1878, the viruses have been pandemic all around the world and the avian influenza has made large number of casualties and huge economic losses.In recent years, the epidemic of H5N1subtype continues to cause a serious impact on global human health and public health security of the world. For example, avian influenza A H5N1virus, which is the first human infection and was reported in Hong Kong in1997and has caused648human infections with384deaths in15countries since2003. Additionally, the H5N1highly pathogenic avian influenza virus outbreak in China Qinghai Lake in2005, which caused more than6,000migratory birds died. The epidemic of H7subtype avian influenza virus caused more than75million poultry deaths and a lot of people infected even lead to death, which outbreak in many countries and regions. A serious epidemic of human infections with a novel avian influenza A (H7N9) virus has emerged in China, the first case of which was recorded in eastern China in February2013. So far, it caused a tragedy that more than100people died.At present, the detection methods of avian influenza virus have a lot, including virology, serology and molecular biology methods, but some are complicated, time-consuming and expensive, so they are not suitable for rapid detection avian influenza virus in primary laboratory, because it is not conducive to monitoring and control the epidemic of avian influenza. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method which is developed based on conventional PCR by Japanese scholar Dr. Notimi T.Its characteristic is thatit uses4or6different primers which specifically designed to recognize6or8distinct regions on the target gene.The LAMP reaction process proceeds at a constant temperature65℃using a DNA polymerase with strand displacement activity. It can be completed in one hour and couldjudge the result only with naked eye. Therefore, it has the potential to replace PCR because of its simplicity, rapidity, specificity and sensitivity, and it doesn’t require expensive laboratory equipment. Therefore, it is very suitable toused in primary laboratory and pen-sites.Based on preliminary study, we applied a new method to judge the results of the amplification products, which observed the fluorescence intensity change by real-time PCR instrument, and optimize the reaction system with this method. We used calcein as fluorescent dye, when positive it will be orange to green. In this paper, we used the conserved sequences of HA and M genes of the H5and H7subtypes avian influenza viruses as our target gene sequences and designed LAMP primers with the PrimerExplorer V4software. We designed two sets LAMP primers of each target gene, and each set LAMP primer comprises outer forward primers F3, out backward primer B3, the forward inner primer FIP, the backward inner primer BIP primers, loop forward primer and loop backward primer. We repeat the test to select the best set primer. The reaction conditions were optimized including reaction temperature, reaction time, Mg2+concentration, dNTP concentration and betaine concentration in LAMP. Then we established optimal LAMP reaction system based on fluorescence intensity change and the width and brightness of gene bands by1.5%agarose gel electrophoresis. The optimal reaction system of M-RT-LAMP is:6mM/LMg2+,1.6mM/L dNTP,0.6M/L betaine,1.6μM/L FIP and BIP,0.2μM/L F3and B3,0.8μM/L LF and LB, optimum temperature63℃. The optimal reaction system of H5-RT-LAMP is:6mM/LMg2+,1.4mM/L dNTP,0.8M/L betaine,1.6μM/L FIP and BIP,0.4μM/L F3and B3,0.8μM/L LF and LB, optimum temperature65℃. The optimal reaction system of H7-RT-LAMP is:6mM/LMg2+,1.4mM/L dNTP,0.8M/L betaine,1.6μM/L FIP and BIP,0.2μM/L F3and B3,0.8μM/L LF and LB, optimum temperature65℃. The fluorescent dye calcein was added to reaction system before experiment, and the reaction system was conducted in optimal temperature and time, then terminate the reaction in80℃for5min. After the reaction, we can judge the result by the fluorescence intensity change, the color change of fluorescence dye and1.5%agarose gel electrophoresis. Using a pool of RNAs extracted from influenza viruses corresponding to some HA subtypes and Newcastle disease virus, the RT-LAMP system was confirmed to amplify only specific AIV RNA. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the real-time fluorescence method was highly sensitive, and that the analytic sensitivity of M-RT-LAMP method is0.01pg; the analytic sensitivity of H5-RT-LAMP method is0.1pg; the analytic sensitivity ofH7-RT-LAMP method is O.lpg.In summary, the RT-LAMP assay established in this study is simple, rapid, sensitive and specific, which can be used as a reliable method for monitoring and early diagnosis of influenza virus infection, especially in underequipped laboratories and pen-sites.