Simultaneous Detection Of Three Arbovirus By Using A One-Step Triplex Rt-Pcr Enzyme Hybridization Assay

Arbovirus is a group of viruses transmitted by infected insect vectors biting sensitive vertebrate.Arbovirus has related to many zoonosis triggering a serious threathen to China public health and livestock breeding. There are various kinds of clinical symptoms will be manifested after infected with arbovirus.Fever and eruption are the most common ones of these manifestations,and it will also produce encephalitis, meningitis even mortality when following a serious onset.Whereas,at present,there is no specific medicine targeting arbovirus and mainly take vaccine inoculation as the necessary prevention step. Therefore, it’s very important to highlight arbovirus effective detection with efficiency to avoid relative diseases eruption and epidemic. Arbovirus detection techniques still has rested on traditional serology test and cell culture for virus isolation in many public healthy organs of China.These techniques are very effective for virus detection and always be called "’Golden Standar" but it’s low sensitivity,comparative long cycle (about one week to get results for cell culture) and lots sample consuming make these techniques hard to adapt to the clinical and field requirements.In this study,we establish a Onestep Triplex RT-PCR Enzyme hybridization Assay which could detect three arbovirus (Japanese Encephallitis virus(JEV),Getah virus (GETV) and Tahyna virus (TAHV)simultaneously.First,allJEV,GETV,andTAHV sequences were downloaded from the public database of the National Center for Biotechnology Information (NCBI)and combined sequences kept in our laboratory were used to design and synthesis primers with biotin modification at5’and specific probes combined with enzyme HRP.Second,proceed onestep RT-PCR assistance by OneStep RT-PCR Kit (QIAGEN), bind the arbovirus RT-PCR product with species specific probe’s and identify the aiming pathogen under a microplate reader after adding HRP substrates. The sensitity of multiplex RT-PCR Enzyme Hybridization in this study was1PFU/mL for JEV,10PFU/mL for GETV and10PFU/mL for TAHV.Apply this method to29CSF samples, results show that strong positive singnal for JEV (the resultant OD value was significant higher than the set cutoff) whereas the other two pathogens (GETV and TAHV) both display negative signals (OD value were significant lower than the set cutoff). Multiplex RT-PCR Enzyme Hybridization has been successfully established in this study which could simultaneously test three arbovirus (JEV,GETV and TAHV).It presents a good sensitivity and speciality with application this method to clinical samples.