Establishment of multiplex suspension array for simultaneous detection of five species of arboviruses / 解放军医学杂志

ABSTRACT

Objective To establish a multiplex suspension array for simultaneously detecting five species of important arbovirus dengue virus (including four serotypes), japanese B encephalitis virus, west nile virus, tick-borne encephalitis virus and yellow fever virus. Method All published genomic sequences of the above arboviruses were collected from GeneBank, and then, the genus universal primers and specific probes were designed according to the conservative and specific NS5 region of the genus Flavivirus. The amine-modified probes were covalently coupled to different carboxylated microspheres by carbodiimide methods. By combining general RT-PCR with virus-specific probe hybridization, a Luminex xMAP system-based suspension array, which could simultaneously differentiate five arboviruses and discriminate 1～4 serotypes of dengue virus, was successfully established. Results A cutoff value twice higher than that of the background fluorescence was judged as positive reaction. RT-PCR products of many arboviruses were used to evaluate the specificity and repeatability of the array, the results showed that the assay was highly specific and had good repeatability. Meanwhile, the coefficient of variability was less than 8% from inter- and intra- assay, implying that both the repeatability and stability of the array were good. Furthermore, several viruses which had been quantitated by plaque assay were used to determine the array’s sensitivity. The results showed that the array’s detection limits were about 1.4 PFU for JBEV and YFV, 14 PFU for WNV respectively. 55 clinical and cultured samples were detected by the established assay, 16 out of the 55 samples were confirmed to be positive. There was only one sample which was not detected contrasted with conventional RT-PCR. However, it had advantages in detecting complex samples rapidly. Conclusion A multiplex suspension array for simultaneously detecting and serotyping five species of important arboviruses has been successfully developed, and it may play an important role in diseases diagnosis and prevention as well as epidemiological studies.