TY - JOUR
T1 - Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles
AU - Chabi, Joseph
AU - Van’t Hof, Arjen
AU - N’dri, Louis K.
AU - Datsomor, Alex
AU - Okyere, Dora
AU - Njoroge, Harun
AU - Pipini, Dimitra
AU - Hadi, Melinda P.
AU - De Souza, Dziedzom K.
AU - Suzuki, Takashi
AU - Dadzie, Samuel K.
AU - Jamet, Helen P.
N1 - Publisher Copyright:
© 2019 Chabi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2019/4
Y1 - 2019/4
N2 - The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72C for An. arabiensis, 75C for An. gambiae and 86C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.
AB - The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72C for An. arabiensis, 75C for An. gambiae and 86C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.
UR - http://www.scopus.com/inward/record.url?scp=85064826564&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0215669
DO - 10.1371/journal.pone.0215669
M3 - Article
C2 - 31002694
AN - SCOPUS:85064826564
SN - 1932-6203
VL - 14
JO - PLoS ONE
JF - PLoS ONE
IS - 4
M1 - e0215669
ER -