Rapid high throughput SYBR green assay for identifying the malaria vectors Anophelese arabiensis, Anopheles coluzzii and Anopheles gambia s.s. Giles

Chabi, Joseph, Van 't Hof, Arjen, N'dri, Louis K., Datsomor, Alex, Okyere, Dora, Njoroge, Harun, Pipini, Dimitra, Hadi, Melinda P., de Souza, Dziedzom K., Suzuki, Takashi, Dadzie, Samuel K. and Jamet, Helen P. (2019) 'Rapid high throughput SYBR green assay for identifying the malaria vectors Anophelese arabiensis, Anopheles coluzzii and Anopheles gambia s.s. Giles'. PLoS ONE, Vol 14, Issue 4, e0215669.

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Abstract

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 72˚C for An. arabiensis, 75˚C for An. gambiae and 86˚C 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.

Item Type:	Article
Subjects:	QX Parasitology > Insects. Other Parasites > QX 515 Anopheles QY Clinical Pathology > QY 4 General works WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
Faculty: Department:	Biological Sciences > Vector Biology Department
Digital Object Identifer (DOI):	https://doi.org/10.1371/journal.pone.0215669
Depositing User:	Stacy Murtagh
Date Deposited:	03 May 2019 13:48
Last Modified:	10 May 2019 15:34
URI:	https://archive.lstmed.ac.uk/id/eprint/10759

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