Abstract
BackgroundInsect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens.MethodsSpecific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 μl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg’s disease virus, and 3000 clinical samples were tested for practicability.ResultsThe optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits.ConclusionsThis optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.
Highlights
Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world
The exogenous RNA fragments packaged by the virus-like particles (VLPs) expression vector pTMSCA2C were used as positive control of bluetongue virus (BTV), epizootic hemorrhagic disease virus of deer (EHDV), West Nile virus (WNV), vesicular stomatitis virus (VSV), Rift Valley fever virus (RVFV), Ebola virus (EBV) and Schmallenberg virus (SBV)
Coupling efficiency of beads and viral probes The capture probes on the breads and the corresponding RC-probes of the 10 pathogens showed a high hybridization signal
Summary
Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. Insect-borne diseases are infectious diseases spread among animals and human through hemophagia by blood-feeding arthropods, such as mosquitoes, ticks and midges, which are widespread in the environment [1]. These infections lead to significant human and animal morbidity and mortality worldwide [1], which cause a huge economic loss. Accurate and timely diagnosis of these infections is a great challenge of utmost importance
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