A new multiplex RT-qPCR method for the simultaneous detection and discrimination of Zika and chikungunya viruses

Abstract

A B S T R A C TObjective: The re-emergence and spread of tropical viruses to new areas has raised a wave of concern worldwide. In order to treat patients at an early stage and prevent the diffusion of an outbreak, early diagnosis, and therefore fast and adequate detection, is needed. To this end, a multiplex reverse transcription real-time polymerase chain reaction TaqMan method was designed to detect Zika (ZIKV) and chikungunya (CHIKV) viruses simultaneously. Methods: Two methods targeting different genome segments were selected from the literature for each virus. These were adapted for high genome coverage and combined in a four-plex assay that was thoroughly validated in-house. The SCREENED tool was used to evaluate the sequence coverage of the method. Results: The full validation approach showed that the new four-plex method allows the specific and sensitive identification and discrimination of ZIKV and CHIKV in routine samples. The combination of two targets per virus allowing almost 100% coverage of about 500 genomes is shown for the first time. Conclusions: PCR is a reliable user-friendly technique that can be applied in remote areas. Such multiplex methods enable early and efficient diagnosis, leading to rapid treatment and effective confinement in outbreak cases. They may also serve as an aid for surveillance, and the full validation permits easy method-transfer allowing worldwide harmonization.

Use of Whole Genome Sequencing Data for a First in Silico Specificity Evaluation of the RT-qPCR Assays Used for SARS-CoV-2 Detection

“…To overcome these limitations, we previously developed an open-access bioinformatics tool named SCREENED (polymeraSe Chain Reaction Evaluation through largE-scale miNing of gEnomic Data [ 39 ]), enabling user-friendly investigations of mismatches between the primers and probes employed in routine RT-qPCR methods, and large amounts of whole genome data, evaluating for each genome in silico the generation of a theoretical RT-qPCR signal. This method has already been successfully used for the evaluation of RT-qPCR tests used for Dengue virus detection [ 39 ], as well as for the Zika and Chikungunya viruses [ 40 ].…”

Internet of medical things (IoMT)-integrated biosensors for point-of-care testing of infectious diseases

“…The similarity of clinical symptoms by these diseases poses a critical clinical challenge. To this end, RT-qPCR method was used by Broeders & team to detect ZIKV and chikungunya (CHIKV) viruses in a single test ( Broeders et al, 2020 ). A new fourplex RT-qPCR assay was thoroughly validated directly for non-invasive samples like urine and saliva.…”

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods

“… Colorimetric format. Electrochemical format Electrochemical format Genosensors R1: 32 pmol L-1 Genosensors R2: 9 pmol L-1 Genosensors R1: 0.7 pmol L-1 Genosensors R2: 3 pmol L-1 [77] Zika virus Chikungunya viruses -a Chikungunya viruses -b Multiplex RT-qPCR 100 copies 5 copies 50 copies [78] Porcine epidemic diarrhea virus Immuno-chromatographic strip 1:50 [79] H1N1 of influenza A virus H3N2 of influenza A virus CdSe/CdS/ZnS quantum dot-linked rapid fluorescent immunochromatographic test 28.37 34.48 [80] Epstein-Barr virus Electrochemical detection 0.46 fM [81] Pseudorabies virus Porcine circovirus 3 SYBR green I-based duplex real-time PCR 37.8 copies/μL, 30.6 copies/μL [82] Classic swine fever virus Porcine circovirus 3 SYBR green I-based duplex real-time fluorescence quantitative PCR 23 copies/μL 36 copies/μL [83] Ebola virus Rolling circle amplification of Ebola virus and fluorescence detection based on graphene oxide 1.4 pM. [84] Ebola virus Microfluidic sample preparation multiplex 0.021 pfu/mL [85] Ebola virus Electrochemical DNA biosensor 4.7nm [86] Ebola virus RT-PCR 10 molecules/...…”