Establishment Of A Multiplex Liquid Chip For Detection Of The Pathogens Of Insect-borne Diseases

Objectives:Insect-borne disease is a class of infectious disease, it is spread by bloodthirsty insects as media. Liquid chip technology is used to establish the detection method of multiple pathogen of insect-borne diseases in the study.Methods:This study was based on the high conserved regions of bluetongue virus (BTV), deer epidemic hemorrhagic virus (EHDV), Q Fever Coxiella burnetii, african swine fever virus (ASFV), west nile virus (WNV), lyme disease Bo burgdorferi (BB), vesicular stomatitis virus (VSV), rift valley fever virus (RVFV), ebola virus (EBV) and schmallenberg virus (SBV), designed 10 pairs of primers and 10 probes. The different encoding magnetic microspheres were effectively coupled with 10 specific probes. The multiple liquid chip system was established for simultaneously detecting 10 insect-borne pathogens after the optimization of key factors including the ratio of the upstream and downstream primers, liquid hybridization temperature and liquid hybridization time. Then the sensitivity-, specificity and repeatability of multiple liquid system were verified.Results:The fragments obtained through PCR/RT-PCR amplification were consistent with the expected.The purified PCR products were linked to carrier to construct positive plasmids.The homology was more than 97% according to DNAMAN analysis. The results showed that the median fluorescence of each insect-borne disease was significantly higher than background values when the proportion of primer was 1:2, the hybridization temperature wsa 50℃ and the hybridization time was 30min. This method can effectively detect target fragments. The results of specificity of multiple liquid system showed the whole system was no cross reaction on similar diseases such as Foot and Mouth disease and peste des petits ruminants. The limit of detection was 102 copies and this method had good repeatability. The insect-borne pathogens mixed samples and 300 nucleic acids of mosquitoes, ticks and midges were tested by the established multiplex vector-borne liquid chip. The detection results of pathogens mixed samples were consistent with known status and 6 Borrelia positive were detected in ticks from Inner Mongolia.Conclusions:The liquid chip method had established successfully in this study for detecting ten pathogens rapidly at the same time. It provided a technology platform for rapid high-throughput screening of Insect-borne diseases. This method will play a vital role for identification and prevention of animal insect-borne diseases.