Table 1 Molecular diagnostic approaches to distinguish members of the An. funestus group

PCR-RFLP-1

PCR primers developed from An. funestus D3 region of 28S ribosomal gene amplified and digested using restriction endonuclease HpaII

An. funestus and An. vaneedeni

Ability to distinguish two morphologically similar species

Limited to only two members of the An. funestus group

Minimal sequence variation between the two species poses the challenge of specificity when other members are added to the assay

Post PCR processing

[48]

Single-strand conformation polymorphism (SSCP) PCR

PCR primers developed from An. funestus D3 region of 28S ribosomal gene amplified and separated based on DNA conformation on SSCP gels

An. funestus, An. vaneedeni, An. rivulorum, and An. leesoni

Ability to distinguish four members of the An. funestus group

Can distinguish east from West African An. funestus

Overlap on the denatured single strand (DSS) banding patterns for An. funestus and An. vaneedeni

Post PCR processing

[49]

Internal transcribed spacer 2 (ITS2)

PCR amplification using internal transcribed spacer 2 (ITS2) region primers

An. funestus and An. rivulorum

Differentiating two sympatric vectors in the An. funestus group

Limited to only two members within the group

Post PCR processing

[50]

ITS2 Cocktail PCR

An. funestus, An. rivulorum, An. vaneedeni, An. leesoni, An. parensis, and An. rivulorum-like

Simultaneous identification of six members within the group in a single PCR run

Post PCR processing

[51, 52]

PCR–RFLP-2

PCR amplification using AFG multiplex PCR assay followed by EcoRI RFLP digestion

An. funestus, An. funestus-like, An. parensis, Anopheles rivulorum, An. vaneedeni, An. leesoni, and An. longipalpis-type C

Identification of An. longipalpis-type C from the other members of the AFG

Post PCR processing and requirement of enzyme digestion

[128]