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Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes.

Faye O, Faye O, Diallo D, Diallo M, Weidmann M, Sall AA - Virol. J. (2013)

Bottom Line: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions.This method was used successfully to detect ZIKV strains from field-caught mosquitoes.We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité des Arbovirus et virus de fièvres hémorragiques, Institut Pasteur Dakar, 36, Avenue Pasteur, BP 220 Dakar, Senegal. asall@pasteur.sn.

ABSTRACT

Background: Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology-which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa.

Results: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes.

Conclusion: We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection.

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Alignment of the designed primers and probe with Zika virus strain sequences. Dots indicate identity with the consensus sequence on the top of the alignment.
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Figure 1: Alignment of the designed primers and probe with Zika virus strain sequences. Dots indicate identity with the consensus sequence on the top of the alignment.

Mentions: An alignment of the NS5 sequences of 13 African and 1 Asian (Malaysia) ZIKV strains identified a highly conserved region of 102 nucleotides (nt) and highly divergent from other flaviviruses. A reverse primer (nt 9352 -TCCRCTCCCYCTYTGGTCTTG-9373), a forward primer (nt 9271- AARTACACATACCARAACAAAGTG GT-9297) and a 16 nt LNA-probe (nt 9304-FAM-CTYAGACCAGCTGAAR-BBQ -9320) were designed (Figure 1) (Table 3).


Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes.

Faye O, Faye O, Diallo D, Diallo M, Weidmann M, Sall AA - Virol. J. (2013)

Alignment of the designed primers and probe with Zika virus strain sequences. Dots indicate identity with the consensus sequence on the top of the alignment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016539&req=5

Figure 1: Alignment of the designed primers and probe with Zika virus strain sequences. Dots indicate identity with the consensus sequence on the top of the alignment.
Mentions: An alignment of the NS5 sequences of 13 African and 1 Asian (Malaysia) ZIKV strains identified a highly conserved region of 102 nucleotides (nt) and highly divergent from other flaviviruses. A reverse primer (nt 9352 -TCCRCTCCCYCTYTGGTCTTG-9373), a forward primer (nt 9271- AARTACACATACCARAACAAAGTG GT-9297) and a 16 nt LNA-probe (nt 9304-FAM-CTYAGACCAGCTGAAR-BBQ -9320) were designed (Figure 1) (Table 3).

Bottom Line: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions.This method was used successfully to detect ZIKV strains from field-caught mosquitoes.We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unité des Arbovirus et virus de fièvres hémorragiques, Institut Pasteur Dakar, 36, Avenue Pasteur, BP 220 Dakar, Senegal. asall@pasteur.sn.

ABSTRACT

Background: Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology-which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa.

Results: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes.

Conclusion: We have developed a rapid, sensitive and specific rRT-PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection.

Show MeSH
Related in: MedlinePlus