Abstract
The sudden and explosive expansion of Zika virus (ZIKV) from the African continent through Oceania and culminating in the outbreak in South America has highlighted the importance of new rapid point-of-care diagnostic tools for the control and prevention of transmission. ZIKV infection has devastating consequences, such as neurological congenital malformations in infants born to infected mothers and Guillain–Barré syndrome in adults. Additionally, its potential for transmission through vector bites, as well as from person to person through blood transfusions and sexual contact, are important considerations for prompt diagnosis. Recombinase polymerase amplification (RPA), an isothermal method, was developed as an alternative field-applicable assay to PCR. Here we report the development of a novel ZIKV real-time reverse transcriptase RPA (RT-RPA) assay capable of detecting a range of different ZIKV strains from a variety of geographical locations. The ZIKV RT-RPA was shown to be highly sensitive, being capable of detecting as few as five copies of target nucleic acid per reaction, and suitable for use with a battery-operated portable device. The ZIKV RT-RPA demonstrated 100 % specificity and 83 % sensitivity in clinical samples. Furthermore, we determined that the ZIKV RT-RPA is a versatile assay that can be applied to crude samples, such as saliva and serum, and can be used as a vector surveillance tool on crude mosquito homogenates. Therefore, the developed ZIKV RT-RPA is a useful diagnostic tool that can be transferred to a resource-limited location, eliminating the need for a specialized and sophisticated laboratory environment and highly trained staff.
Highlights
Zika virus (ZIKV) is a positive-sense single-stranded RNA virus, a member of the genus flavivirus, family Flaviviridae, with a genome of ~10.2 kb
The template region amplified in the ZIKV RT-recombinase polymerase amplification (RPA) was selected based on its high level of sequence conservation, as determined by the sequence alignment of strains isolated from different outbreaks (Fig. 1)
The strain MP1751 (NCBI accession number KY288905) originated from Uganda, Africa, in 1962, and its temporal and geographical distance from the rest of the ZIKV strains is reflected in the greater sequence variation of its ZIKV reverse transcriptase RPA (RT-RPA) target region, with the reverse primer differing by three base pairs and the probe by five base pairs from the MP1751 ZIKV strain target region (Table 1) [68]
Summary
Zika virus (ZIKV) is a positive-sense single-stranded RNA virus, a member of the genus flavivirus, family Flaviviridae, with a genome of ~10.2 kb. It is divided into two known lineages, namely the African and Asian [1]. ZIKV research is developing rapidly and new guidance on prevention, diagnosis and surveillance is constantly being shaped, as new data become available [43]. Other arboviruses, such as those that cause dengue and chikungunya, are transmitted by Aedes mosquitoes. As these have similar clinical manifestations, an accurate diagnosis is important for patient management, relevant medical advice, epidemiological follow-up, contact tracing and vector control operations
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