Assay optimization for molecular detection of Zika virus

Abstract

ObjectiveTo examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection.MethodsWe compared seven published real-time RT–PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we constructed a synthetic universal control ribonucleic acid (uncRNA) containing all of the assays’ target regions on one RNA strand and spiked human blood or urine with known quantities of African or Asian Zika virus strains. Viral loads in 33 samples from Zika virus-infected patients were determined by using one of the new assays.FindingsOligonucleotides of the published real-time RT–PCR assays, showed up to 10 potential mismatches with the Asian lineage causing the current outbreak, compared with 0 to 4 mismatches for the new assays. The 95% lower detection limit of the seven most sensitive assays ranged from 2.1 to 12.1 uncRNA copies/reaction. Two assays had lower sensitivities of 17.0 and 1373.3 uncRNA copies/reaction and showed a similar sensitivity when using spiked samples. The mean viral loads in samples from Zika virus-infected patients were 5 × 104 RNA copies/mL of blood and 2 × 104 RNA copies/mL of urine.ConclusionWe provide reagents and updated protocols for Zika virus detection suitable for the current outbreak strains. Some published assays might be unsuitable for Zika virus detection, due to the limited sensitivity and potential incompatibility with some strains. Viral concentrations in the clinical samples were close to the technical detection limit, suggesting that the use of insensitive assays will cause false-negative results.

Establishment and cryptic transmission of Zika virus in Brazil and the Americas

“…The American ZIKV epidemic comprises a single founder lineage 4,22,23 (hereafter termed Am-ZIKV) derived from Asian genotype viruses (hereafter termed PreAm-ZIKV) from southeast Asia and the Pacific 4 . A sliding window analysis of pairwise genetic diversity along the ZIKV genome shows that the diversity of PreAm-ZIKV strains is on average about two-fold greater than that of Am-ZIKV viruses (Fig.…”

Zika virus RNA polymerase chain reaction on the utility channel of a commercial nucleic acid testing system

“…Therefore, it is difficult to compare our assay with previously published ZIKV PCR assays. However, a recent study compared nine published ZIKV PCR assays using a synthetic universal control RNA construct containing the target regions of each assay . Extrapolation of the findings of this study to viral loads and accommodating the loads to our input and extraction volume, the LOD of eight assays out of nine ranged between 20 and 170 copies/mL.…”

Pathogenic viruses: Molecular detection and characterization

“…Therefore, a reliable assay, such as molecular detection of viral nucleic acid, is required to detect Zika virus in infected people. The genomics domain commonly targeted for molecular detection of this virus include the membrane (M), envelope (E), nonstructural proteins (NS) 1, NS2B, NS3 and NS5 genes (Corman et al, 2016;Perkasa et al, 2016).…”