| Zika Virus(ZIKV),which belongs to the genus of Flavivirus within the Flaviviridae family,is a positive-sense,single-stranded RNA virus.In 2015,the outbreak of ZIKV in Brazil caused millions of people to be infected,which had aroused great concern and vigilance from health departments in various countries around the world.ZIKV infection is always associated with neurological and autoimmune complications,such as prenatal microcephaly(PIM)and Guillain-Barre syndrome(GBS),which cause a severe disease burden on the local population.In addition,the ZIKV has the same transmission media as Dengue virus(DENV),Yellow fever virus(YFV)and Chikungunya virus(CHIKV)and the clinical symptoms caused by these viruses are similar,which makes it difficult to distinguish ZIKV infection at early stage.However,early diagnosis facilitates identification of viral infection and controls the spread of the disease.Therefore,sensitive and specific detection reagents that could distinguish these pathogens are urgently needed for the prevention and control of ZIKV.At present,there are three main diagnostic methods for ZIKV:cell-culture isolation,serological test and real time RT-PCR(qRT-PCR).Cell-cuture isolation is the gold standard for ZIKV detection and identification,but not suitable for the early diagnosis since it is time-consuming and laborious.Serological tests mainly include ELISAs and Neutralization tests(NTs).In the early clinical stage of ZIKV infection,the level of serologic IgM is quite low,which may lead to a false-negative result,.Besides,the substantial cross-reactivity among the four viruses might results in false-positive results.Therefore,serological test is not the preferred method in the detection of ZIKV.The qRT-PCR method has been widely used in the diagnosis of these viruses because of the characteristics of rapid,sensitive and specific.However,a multiplex real time RT-PCR capable of detecting these four viruses simultaneously has not been reported yet.Therefore,we seek to establish a multiplex qRT-PCR reagent for the rapid detection and identification of these four viruses.The full genome sequences of these four viruses were downloaded from GenBank and aligned using the Clustal W method to determine the specific and conserved regions in MEGA5.0.Then the primers and probes were designed within the conserved regions.Using the commercial reagents as controls,we developed four singleplex qRT-PCRs against the corresponding virus.Based on the DENV singleplex qRT-PCR,a duplex qRT-PCR for DENV and CHIKV,a triplex qRT-PCR for DENV,CHIKV and ZIKV,and a final quadruple qRT-PCR were developed successively.After the opitimization of the reaction system and amplification conditions,the performance of the quadruplex qRT-PCR assay was evaluated.The 95%lower limit of detection(LLOD)of the quadruplex qRT-PCR was about 11-32 copies/test.The linear range was determined and the R2 value for each virus was>0.98.In the detection of overlapping infections with different viral loads,the Ct value obtained by the quadruplex qRT-PCR for each virus was not significantly different from the Ct value obtained from the corresponding singleplex qRT-PCR(P= 0.26>0.05).In the simulation of ZIKV clinical specimens,the result of our quadruplex qRT-PCR was similar to that detected by the commercial singleplex qRT-PCR assay and the bias(difference between the means of Ct)was only 0.08(95%CI:-0.68?0.84).In addition,the specificity of our quadruplex qRT-PCR was up to 100%,and the coefficiency of variation of RNA templates with different concentrations was<3%,which indicated good stability of our quadruplex assay.In summary,a quadruplex real time RT-PCR system was established and it could offer quick and effective differential diagnosis of ZIKV,DENV,YFV and CHIKV in a single tube.It is beneficial to the early treatment of diseases and the control of the epidemic,providing an important guarantee for the prevention and control of ZIKV. |
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