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Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex
Acta Tropica 107 (2008) 50–53
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex Chris Bass ∗ , Martin S. Williamson, Linda M. Field Centre for Sustainable Pest and Disease Management, Biological Chemistry Department, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK
a r t i c l e
i n f o
Article history: Received 25 February 2008 Received in revised form 1 April 2008 Accepted 7 April 2008 Available online 15 April 2008 Keywords: Anopheles gambiae Species complex Real-time PCR
a b s t r a c t Two high-throughput assays for the identification of members of the Anopheles gambiae sensu lato species complex have recently been reported. These methods, are based on TaqMan single nucleotide polymorphism (SNP) genotyping that enables rapid scoring of mosquito DNA samples in real-time PCR reactions. Unfortunately, both assays are restricted in the number of species that they can identify and a combination of the two assays may be required to identify all possible species in certain regions. To overcome this limitation, and thereby further increase throughput while reducing costs, we have developed a new multiplex real-time PCR assay for identifying members of the An. gambiae complex. The new method uses three probes labelled with fluorophores with distinct emission and excitation spectra, allowing simultaneous detection of the two main malaria vectors from the non-vector sibling species, and can be used on single mosquito legs from silica-dried specimens. A genotyping trial of over 450 specimens collected from 13 countries in sub-Saharan Africa showed the multiplex assay to be highly specific and sensitive and it compared well against the two previously reported TaqMan assays and standard allele-specific PCR. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The Anopheles gambiae sensu lato (s.l.) species complex comprises seven sibling species of mosquitoes and includes the most important vectors of malaria worldwide. The members of this complex are morphologically indistinguishable but differ in their behaviour and ability to vector malaria. An. gambiae s.s. and Anopheles arabiensis are the two principal malaria vectors, whilst two other members of the complex, Anopheles melas and Anopheles merus, are potential vectors but their effect is limited by their distribution which is confined to coastal regions because of their requirement for brackish water as breeding sites (Tsy et al., 2003; Moreno et al., 2004). Similarly Anopheles bwambae has an extremely limited distribution close to the Buranga hot springs in Uganda and therefore has only local importance as a vector (White, 1985). Anopheles quadriannulatus species A, which is widespread in southern Africa, and An. quadriannulatus species B, found in Ethiopia, are considered to be zoophilic non-malaria vectors (Coetzee, 2004). Thus for vector control strategies and ecological research there is often a need to identify the two principal vector species An. gambiae s.s. and
∗ Corresponding author. Tel.: +44 1582 763133. E-mail address: [email protected] (C. Bass). 0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.04.009
An. arabiensis and to distinguish them from other members of the complex. To this end a number of molecular species identification assays have been developed, many of which exploit species-specific polymorphisms in the ribosomal DNA gene (Scott et al., 1993; Favia et al., 1997; Fanello et al., 2002) and recently two high-throughput methods based on real-time PCR have been described (Bass et al., 2007; Walker et al., 2007). These recent assays are based on TaqMan single nucleotide polymorphism (SNP) genotyping and are ‘closed tube’ approaches that require only a single step to characterise a mosquito DNA sample. The first of these methods (referred to as the Walker TaqMan assay in this manuscript) detects and discriminates between An. gambiae s.s. and An. arabiensis but is unsuitable for testing other members of the complex as the assay incorrectly identifies the three species An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The second TaqMan assay (referred to as the Bass TaqMan assay in this manuscript) identifies An. gambiae s.s. and An. arabiensis as one group and An. quadriannulatus, An. melas or An. merus as a second group. The two assays can be used sequentially, with the Bass assay run first and samples that are scored as An. gambiae s.s./An. arabiensis further identified to species using the Walker TaqMan assay. Here we describe the development of a new multiplex real-time PCR assay that requires only a single reaction to detect and discriminate An. gambiae s.s. from An. arabiensis and An. quadriannulatus/An. melas/An. merus.
C. Bass et al. / Acta Tropica 107 (2008) 50–53
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2. Materials and methods
2.2. Multiplex real-time PCR assay
2.1. Preparation of mosquito samples for species identification trial
The multiplex real-time PCR assay was based on the same region of the rRNA gene, and employed the same standard forward and reverse primers, as the Walker TaqMan assay (Walker et al., 2007). Three probes were designed within the region encompassed by these primers. Two of these were TaqMan minor groove binding (MGB) probes (Applied Biosystems, Foster City, CA) designed to detect either An. gambiae s.s. (probe Ag 5 TGGAGCGGaACAC-3 ) or An. quadriannulatus/An. melas/An. merus (probe Aq 5 -TGGAGCGGgACAC-3 ) by exploiting a single polymorphism between these species. MGB probes provide more accurate allelic discrimination by increasing the TM between matched and mis-matched probes (Afonina et al., 1997). The final probe (probe Aa 5 -AC + A + T + AG + GATGGA + G + A + AGG-3 ) was designed to detect An. arabiensis and employed locked nucleic acid (LNA) modified nucleotides (Sigma Aldrich, St. Louis, MO) which have a similar effect to the MGB moiety (Simeonov and Nikiforov, 2002). The three probes were labelled at the 5 end with VIC (probe Ag) for An. gambiae s.s. detection, 6FAM (probe Aq) for An. quadriannulatus/An. melas/An. merus detection and Cy5 (probe Aa) for An. arabiensis detection. These three flurophores have distinct emission and excitation spectra allowing their independent detection in a single reaction. Multiplex PCR reactions (25 l) contained 1–2 l of genomic DNA, 12.5 l of SensiMix DNA kit (Quantace, Norwood, MA),
In order to directly compare the multiplex real-time PCR assay with the two previously reported TaqMan assays and standard allele-specific PCR (AS-PCR) the same collection of mosquito DNA samples used in the study by Bass et al. (2007) was also used to optimise and test the assay developed in this study. For the blind genotyping trial the 466 sample set was randomly pipetted into five new 96-well test plates. The samples had originally been collected from the field in Cameroon, Ghana, Kenya, South Africa, Malawi, Sao Tome, La Reunion, Tanzania, Sudan, Angola, Burkina Faso, Gabon, and Mozambique and their species determined at the time of collection using the standard allele-specific PCR method (Scott et al., 1993). The samples included 169 An. gambiae s.s. (a mixture of M and S molecular forms), 173 An. arabiensis, 66 An. quadriannulatus and 21 samples of An. melas/An. merus; the remaining samples were either undetermined or negative controls. To determine the sensitivity of the new method a dilution series of standard DNA samples from each of the five main species was included. For this, the DNAs were diluted to 20 ng/l (as determined by absorption at 260 nm using a NanoDrop spectrophotometer, NanoDrop Technologies, Wilmington, DE) and then serial dilutions were made down to a 1 in 1 × 106 .
Fig. 1. Species identification using the multiplex real-time PCR assay. Twenty or more specimens of An. gambiae s.s. (red trace), An. arabiensis (blue trace) and An. quadriannulatus/An. melas/An. merus (green trace) were tested. (A) Cycling of probe Ag (VIC-labelled) specific to An. gambiae s.s. (B) Cycling of probe Aa (Cy5-labelled) specific to An. arabiensis. (C) Cycling of probe Aq (6FAM-labelled) specific to An. quadriannulatus/An. melas/An. merus.(For interpretation of the reference to colour in this figure the reader is referred to the web version of this article)
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C. Bass et al. / Acta Tropica 107 (2008) 50–53
800 nM of each primer, 200 nM of probes Aa and Aq and 80 nM of probe Ag. Samples were run on a Rotor-Gene 6000TM (Corbett Research, Sydney, Australia) using temperature cycling conditions of 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 25 s and 66 ◦ C for 60 s. The increases in VIC, FAM and Cy5 fluorescence were monitored in real time by acquiring each cycle on the yellow (530 nm excitation and 555 nm emission) green (470 nm excitation and 510 emission) and red (625 nm excitation and 660 nm emission) channels of the Rotor-Gene respectively. Amplification reactions on whole mosquitoes, or single legs were done by placing the mosquito or leg in the reaction tube and simply covering with the PCR mastermix. For these experiments whole mosquitoes or single legs of 10 specimens each of An. arabiensis, An. quadriannulatus and An. gambiae were tested. The An. gambiae specimens were obtained from the laboratory colonies, Kisumu and RSP. The An. arabiensis and An. quadriannulatus samples were field-collected mosquitoes from Malawi that had been stored on silica and previously identified using a single leg as template by the standard AS-PCR (Scott et al., 1993).
12 (2.57%) failed samples (as compared to 1.25% and 2.96% for the Bass and Walker TaqMan assay and 15% for AS-PCR using the same samples set). The analytical sensitivity of the assay was examined using a dilution series of DNAs from each of the five species, both as part of the blind trial and also subsequently with dilutions of two additional DNA templates. The limit of detection for each species was between a 1 in 1600 and 1 in 10,000 dilution representing a low limit detection of 2–12.5 pg DNA. We also carried out additional experiments to investigate the possibility of using whole mosquitoes or single mosquito legs in PCR instead of genomic DNA. In this case, the legs or bodies were placed in the PCR tube and covered with the PCR reaction mix. When whole mosquito bodies were used as template for PCR a high level of background fluorescence was observed which prevented unambiguous scoring, however using single legs from fresh or silica-dried mosquitoes was found to work nearly as well as using extracted DNA. Ten from ten An. gambiae and An. arabiensis leg samples and nine from ten An. quadriannultaus leg samples were correctly scored using this approach with a single failed reaction.
3. Results
4. Discussion
Initial experiments optimised the new real-time PCR assay as three singleplex reactions (each probe used in a separate PCR reaction) on control DNA templates of known Anopheles species. Using an annealing/extension time of 60 ◦ C the probes Aa and Aq (Cy5and 6FAM-labelled) showed specific amplification of An. arabiensis and An. quadriannulatus/An. melas/An. merus respectively. At this temperature the probe Ag (VIC-labelled) also gave sensitive detection of An. gambiae s.s., however, when An. quadriannulatus, An. melas or An. merus DNAs were tested a low level ‘background’ fluorescence signal was observed, presumably from non-specific binding of this probe (data not shown). This could be eliminated by increasing the annealing/extension temperature to 66 ◦ C and lowering the final probe concentration in the PCR from 200 to 80 nM. The three probes were combined in a multiplex assay (using the increased annealing/extension) temperature and no loss of sensitivity or specificity was observed. An example of using the assay to test 20 or more samples of each species is shown in Fig. 1. A substantial increase in Cy5 fluorescence (probe Aa) during PCR identifies an An. arabiensis specimen, an increase in VIC fluorescence (probe Ag) identifies an An. gambiae s.s. sample, and an increase in 6FAM fluorescence (probe Aq) identifies An. quadriannulatus, An. melas or An. merus specimen. An increase in two or more of the dyes would indicate a hybrid or a contaminated sample. The new multiplex assay was then used to genotype the test samples which had been previously used to test the Bass and Walker TaqMan assays and AS-PCR and a comparison of the results is given in Table 1. The new multiplex real-time PCR assay shows a high level of specificity with only two incorrect identifications (0.43%). In both cases the samples were scored as ‘hybrids’ (An. gambiae s.s./An. arabiensis and An. arabiensis/An. quadriannulatus). This result may have been due to a non-specific signal from one of the fluorescent probes or from DNA contamination during the setting up of the PCRs. The new assay also shows only a low level of ‘failed’ reactions in the trial with
The rapid identification of vector and non-vector species of the An. gambiae complex is often an important part of vector control strategies. Two high-throughput TaqMan assays have recently been developed for this purpose and both were shown to be significantly more sensitive than standard PCR (Bass et al., 2007; Walker et al., 2007). However, both assays have limitations. The Walker TaqMan assay is designed to detect and discriminate only An. gambiae s.s. and An. arabiensis and is unsuitable for testing other members of the complex (the assay incorrectly identifies An. quadriannulatus, An. merus and An. melas as An. gambiae s.s.). The Bass TaqMan assay distinguishes members of the complex in two groups, the main malaria vectors An. gambiae s.s. and An. arabiensis as one group and An. quadriannulatus, An. melas and An. merus as a second group. Used alone, the Bass TaqMan assay is unable to discriminate between An. gambiae s.s. and An. arabiensis which may be significant for certain studies, such as those examining the spread of resistance genes in mosquito populations. To overcome this, the two TaqMan assays can be used sequentially to further identify samples that are scored as An. gambiae s.s./An. arabiensis in the Bass TaqMan by subsequent testing using the Walker TaqMan. However, this has the obvious disadvantage of both increasing costs and lowering throughput. The new multiplex real-time PCR assay described here overcomes this by using three probes labelled with fluorophores with distinct emission and excitation spectra allowing sensitive and specific detection of (1) An. gambiae s.s. (2) An. arabiensis and (3) An. quadriannulatus/An. melas/An. merus as a group. Unfortunately, extensive examination of the available rRNA gene sequences did not reveal a suitable position to site additional probes that would allow the discrimination of An. quadriannulatus from An. melas and An. merus by this method. However, although An. melas and An. merus have been shown to be malaria vectors their distribution is limited to costal regions. In contrast, An. quadriannulatus species A is a nonvector species and is widespread in southern Africa where it is often
Table 1 Comparison of the genotyping results of the new multiplex PCR assay with data obtained previously by three alternative methods (Bass et al., 2007) Genotyping results
Correct scores Failed reactions Failed reactions excluding dilution series Incorrect scores
Bass TaqMan
AS-PCR
Walker TaqMan
Multiplex assay
455/466 11 5 0
364/466 99 60 3
333/371 25 11 2
421/466 31 12 2
C. Bass et al. / Acta Tropica 107 (2008) 50–53
sympatric with An. gambiae s.s. and/or An. arabiensis. Thus the new multiplex assay has application as (i) a vector/non-vector species identification test for large parts of sub-Saharan Africa (except for coastal regions) (ii) a means of distinguishing An. melas from An. gambiae s.s./An. arabiensis where they are sympatric such as regions along the west coast of Africa. The latter is possible because An. melas does not occur sympatrically with An. merus or An. quadriannulatus (Coetzee et al., 2000). It should, however, be used with caution in regions close to the east coast of Africa where An. quadriannulatus has been found to occur sympatrically with An. merus (Coetzee et al., 2000). Like the Bass TaqMan assay the new method will also correctly identify a ‘failed’ reaction as all members of the complex (except for An. bwambae which was not tested due to its rarity) are detected by one of the three fluorescent-labelled probes. The cost of the new multiplex PCR method is in the region of US $ 1 per mosquito analysed which represents a significant reduction in the cost of running the Bass and Walker TaqMan assays sequentially which costs in the region of US $ 1.5. The results of the genotyping trial carried out in this study showed that multiplexing the realtime PCRs did not result in any loss of sensitivity or specificity with 97% of samples correctly scored (compared to 96.5% and 98.75% for the Walker and Bass TaqMan assays using the same sample set). In addition, mosquitoes can be assigned to a species using a single leg without the need to first extract DNA, which increases throughput and allows further diagnostics (such as detection of the malaria parasite) to be conducted on the remaining tissue if required. Acknowledgements We thank Craig Wilding and Martin Donnelly (Liverpool School of Tropical Medicine) for collecting and organising the original mosquito samples. This work was supported by the Innovative Vector Control Consortium. Rothamsted Research receives grant-aided
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support from the Biotechnology and Biological Research Council of the United Kingdom. References Afonina, I., Zivarts, M., Kutyavin, I., Lukhtanov, E., Gamper, H., Meyer, R.B., 1997. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25, 2657–2660. Bass, C., Williamson, M.S., Wilding, C.S., Donnelly, M.J., Field, L.M., 2007. Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay. Malar. J. 6, 155. Coetzee, M., 2004. Distribution of the African malaria vectors of the Anopheles gambiae complex. Am. J. Trop. Med. Hyg. 70, 103–104. Coetzee, M., Craig, M., le Sueur, D., 2000. Distribution of African malaria mosquitoes belonging to the Anopheles gambiae complex. Parasitol. Today 16, 74–77. Fanello, C., Santolamazza, F., della Torre, A., 2002. Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR–RFLP. Med. Vet. Entomol. 16, 461–464. Favia, G., Della Torre, A., Bagayoko, M., Lanfrancotti, A., Sagnon, N., Toure, Y.T., Coluzzi, M., 1997. Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation. Insect Mol. Biol. 6, 377–383. Moreno, M., Cano, J., Nzambo, S., Bobuakasi, L., Buatiche, J.N., Ondo, M., Micha, F., Benito, A., 2004. Malaria panel assay versus PCR: detection of naturally infected Anopheles melas in a coastal village of equatorial Guinea. Malar. J. 3, 20. Scott, J.A., Brogdon, W.G., Collins, F.H., 1993. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am. J. Trop. Med. Hyg. 49, 520–529. Simeonov, A., Nikiforov, T.T., 2002. Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. Nucleic Acids Res. 30, e91. Tsy, J., Duchemin, J.B., Marrama, L., Rabarison, P., Le Goff, G., Rajaonarivelo, V., Robert, V., 2003. Distribution of the species of the Anopheles gambiae complex and first evidence of Anopheles merus as a malaria vector in Madagascar. Malar. J. 2, 33. Walker, E.D., Thibault, A.R., Thelen, A.P., Bullard, B.A., Huang, J., Odiere, M.R., Bayoh, N.M., Wilkins, E.E., Vulule, J.M., 2007. Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping. Malar. J. 6, 23. White, G.B., 1985. Anopheles bwambae sp. n., a malaria vector in Semliki Valley, Uganda, and its relationships with other sibling species of the An. gambiae complex. Syst. Entomol. 10, 501–522.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Development of a multiplex real-time PCR assay for identification of members of the Anopheles gambiae species complex Chris Bass ∗ , Martin S. Williamson, Linda M. Field Centre for Sustainable Pest and Disease Management, Biological Chemistry Department, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK
a r t i c l e
i n f o
Article history: Received 25 February 2008 Received in revised form 1 April 2008 Accepted 7 April 2008 Available online 15 April 2008 Keywords: Anopheles gambiae Species complex Real-time PCR
a b s t r a c t Two high-throughput assays for the identification of members of the Anopheles gambiae sensu lato species complex have recently been reported. These methods, are based on TaqMan single nucleotide polymorphism (SNP) genotyping that enables rapid scoring of mosquito DNA samples in real-time PCR reactions. Unfortunately, both assays are restricted in the number of species that they can identify and a combination of the two assays may be required to identify all possible species in certain regions. To overcome this limitation, and thereby further increase throughput while reducing costs, we have developed a new multiplex real-time PCR assay for identifying members of the An. gambiae complex. The new method uses three probes labelled with fluorophores with distinct emission and excitation spectra, allowing simultaneous detection of the two main malaria vectors from the non-vector sibling species, and can be used on single mosquito legs from silica-dried specimens. A genotyping trial of over 450 specimens collected from 13 countries in sub-Saharan Africa showed the multiplex assay to be highly specific and sensitive and it compared well against the two previously reported TaqMan assays and standard allele-specific PCR. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The Anopheles gambiae sensu lato (s.l.) species complex comprises seven sibling species of mosquitoes and includes the most important vectors of malaria worldwide. The members of this complex are morphologically indistinguishable but differ in their behaviour and ability to vector malaria. An. gambiae s.s. and Anopheles arabiensis are the two principal malaria vectors, whilst two other members of the complex, Anopheles melas and Anopheles merus, are potential vectors but their effect is limited by their distribution which is confined to coastal regions because of their requirement for brackish water as breeding sites (Tsy et al., 2003; Moreno et al., 2004). Similarly Anopheles bwambae has an extremely limited distribution close to the Buranga hot springs in Uganda and therefore has only local importance as a vector (White, 1985). Anopheles quadriannulatus species A, which is widespread in southern Africa, and An. quadriannulatus species B, found in Ethiopia, are considered to be zoophilic non-malaria vectors (Coetzee, 2004). Thus for vector control strategies and ecological research there is often a need to identify the two principal vector species An. gambiae s.s. and
∗ Corresponding author. Tel.: +44 1582 763133. E-mail address: [email protected] (C. Bass). 0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.04.009
An. arabiensis and to distinguish them from other members of the complex. To this end a number of molecular species identification assays have been developed, many of which exploit species-specific polymorphisms in the ribosomal DNA gene (Scott et al., 1993; Favia et al., 1997; Fanello et al., 2002) and recently two high-throughput methods based on real-time PCR have been described (Bass et al., 2007; Walker et al., 2007). These recent assays are based on TaqMan single nucleotide polymorphism (SNP) genotyping and are ‘closed tube’ approaches that require only a single step to characterise a mosquito DNA sample. The first of these methods (referred to as the Walker TaqMan assay in this manuscript) detects and discriminates between An. gambiae s.s. and An. arabiensis but is unsuitable for testing other members of the complex as the assay incorrectly identifies the three species An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The second TaqMan assay (referred to as the Bass TaqMan assay in this manuscript) identifies An. gambiae s.s. and An. arabiensis as one group and An. quadriannulatus, An. melas or An. merus as a second group. The two assays can be used sequentially, with the Bass assay run first and samples that are scored as An. gambiae s.s./An. arabiensis further identified to species using the Walker TaqMan assay. Here we describe the development of a new multiplex real-time PCR assay that requires only a single reaction to detect and discriminate An. gambiae s.s. from An. arabiensis and An. quadriannulatus/An. melas/An. merus.
C. Bass et al. / Acta Tropica 107 (2008) 50–53
51
2. Materials and methods
2.2. Multiplex real-time PCR assay
2.1. Preparation of mosquito samples for species identification trial
The multiplex real-time PCR assay was based on the same region of the rRNA gene, and employed the same standard forward and reverse primers, as the Walker TaqMan assay (Walker et al., 2007). Three probes were designed within the region encompassed by these primers. Two of these were TaqMan minor groove binding (MGB) probes (Applied Biosystems, Foster City, CA) designed to detect either An. gambiae s.s. (probe Ag 5 TGGAGCGGaACAC-3 ) or An. quadriannulatus/An. melas/An. merus (probe Aq 5 -TGGAGCGGgACAC-3 ) by exploiting a single polymorphism between these species. MGB probes provide more accurate allelic discrimination by increasing the TM between matched and mis-matched probes (Afonina et al., 1997). The final probe (probe Aa 5 -AC + A + T + AG + GATGGA + G + A + AGG-3 ) was designed to detect An. arabiensis and employed locked nucleic acid (LNA) modified nucleotides (Sigma Aldrich, St. Louis, MO) which have a similar effect to the MGB moiety (Simeonov and Nikiforov, 2002). The three probes were labelled at the 5 end with VIC (probe Ag) for An. gambiae s.s. detection, 6FAM (probe Aq) for An. quadriannulatus/An. melas/An. merus detection and Cy5 (probe Aa) for An. arabiensis detection. These three flurophores have distinct emission and excitation spectra allowing their independent detection in a single reaction. Multiplex PCR reactions (25 l) contained 1–2 l of genomic DNA, 12.5 l of SensiMix DNA kit (Quantace, Norwood, MA),
In order to directly compare the multiplex real-time PCR assay with the two previously reported TaqMan assays and standard allele-specific PCR (AS-PCR) the same collection of mosquito DNA samples used in the study by Bass et al. (2007) was also used to optimise and test the assay developed in this study. For the blind genotyping trial the 466 sample set was randomly pipetted into five new 96-well test plates. The samples had originally been collected from the field in Cameroon, Ghana, Kenya, South Africa, Malawi, Sao Tome, La Reunion, Tanzania, Sudan, Angola, Burkina Faso, Gabon, and Mozambique and their species determined at the time of collection using the standard allele-specific PCR method (Scott et al., 1993). The samples included 169 An. gambiae s.s. (a mixture of M and S molecular forms), 173 An. arabiensis, 66 An. quadriannulatus and 21 samples of An. melas/An. merus; the remaining samples were either undetermined or negative controls. To determine the sensitivity of the new method a dilution series of standard DNA samples from each of the five main species was included. For this, the DNAs were diluted to 20 ng/l (as determined by absorption at 260 nm using a NanoDrop spectrophotometer, NanoDrop Technologies, Wilmington, DE) and then serial dilutions were made down to a 1 in 1 × 106 .
Fig. 1. Species identification using the multiplex real-time PCR assay. Twenty or more specimens of An. gambiae s.s. (red trace), An. arabiensis (blue trace) and An. quadriannulatus/An. melas/An. merus (green trace) were tested. (A) Cycling of probe Ag (VIC-labelled) specific to An. gambiae s.s. (B) Cycling of probe Aa (Cy5-labelled) specific to An. arabiensis. (C) Cycling of probe Aq (6FAM-labelled) specific to An. quadriannulatus/An. melas/An. merus.(For interpretation of the reference to colour in this figure the reader is referred to the web version of this article)
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C. Bass et al. / Acta Tropica 107 (2008) 50–53
800 nM of each primer, 200 nM of probes Aa and Aq and 80 nM of probe Ag. Samples were run on a Rotor-Gene 6000TM (Corbett Research, Sydney, Australia) using temperature cycling conditions of 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 25 s and 66 ◦ C for 60 s. The increases in VIC, FAM and Cy5 fluorescence were monitored in real time by acquiring each cycle on the yellow (530 nm excitation and 555 nm emission) green (470 nm excitation and 510 emission) and red (625 nm excitation and 660 nm emission) channels of the Rotor-Gene respectively. Amplification reactions on whole mosquitoes, or single legs were done by placing the mosquito or leg in the reaction tube and simply covering with the PCR mastermix. For these experiments whole mosquitoes or single legs of 10 specimens each of An. arabiensis, An. quadriannulatus and An. gambiae were tested. The An. gambiae specimens were obtained from the laboratory colonies, Kisumu and RSP. The An. arabiensis and An. quadriannulatus samples were field-collected mosquitoes from Malawi that had been stored on silica and previously identified using a single leg as template by the standard AS-PCR (Scott et al., 1993).
12 (2.57%) failed samples (as compared to 1.25% and 2.96% for the Bass and Walker TaqMan assay and 15% for AS-PCR using the same samples set). The analytical sensitivity of the assay was examined using a dilution series of DNAs from each of the five species, both as part of the blind trial and also subsequently with dilutions of two additional DNA templates. The limit of detection for each species was between a 1 in 1600 and 1 in 10,000 dilution representing a low limit detection of 2–12.5 pg DNA. We also carried out additional experiments to investigate the possibility of using whole mosquitoes or single mosquito legs in PCR instead of genomic DNA. In this case, the legs or bodies were placed in the PCR tube and covered with the PCR reaction mix. When whole mosquito bodies were used as template for PCR a high level of background fluorescence was observed which prevented unambiguous scoring, however using single legs from fresh or silica-dried mosquitoes was found to work nearly as well as using extracted DNA. Ten from ten An. gambiae and An. arabiensis leg samples and nine from ten An. quadriannultaus leg samples were correctly scored using this approach with a single failed reaction.
3. Results
4. Discussion
Initial experiments optimised the new real-time PCR assay as three singleplex reactions (each probe used in a separate PCR reaction) on control DNA templates of known Anopheles species. Using an annealing/extension time of 60 ◦ C the probes Aa and Aq (Cy5and 6FAM-labelled) showed specific amplification of An. arabiensis and An. quadriannulatus/An. melas/An. merus respectively. At this temperature the probe Ag (VIC-labelled) also gave sensitive detection of An. gambiae s.s., however, when An. quadriannulatus, An. melas or An. merus DNAs were tested a low level ‘background’ fluorescence signal was observed, presumably from non-specific binding of this probe (data not shown). This could be eliminated by increasing the annealing/extension temperature to 66 ◦ C and lowering the final probe concentration in the PCR from 200 to 80 nM. The three probes were combined in a multiplex assay (using the increased annealing/extension) temperature and no loss of sensitivity or specificity was observed. An example of using the assay to test 20 or more samples of each species is shown in Fig. 1. A substantial increase in Cy5 fluorescence (probe Aa) during PCR identifies an An. arabiensis specimen, an increase in VIC fluorescence (probe Ag) identifies an An. gambiae s.s. sample, and an increase in 6FAM fluorescence (probe Aq) identifies An. quadriannulatus, An. melas or An. merus specimen. An increase in two or more of the dyes would indicate a hybrid or a contaminated sample. The new multiplex assay was then used to genotype the test samples which had been previously used to test the Bass and Walker TaqMan assays and AS-PCR and a comparison of the results is given in Table 1. The new multiplex real-time PCR assay shows a high level of specificity with only two incorrect identifications (0.43%). In both cases the samples were scored as ‘hybrids’ (An. gambiae s.s./An. arabiensis and An. arabiensis/An. quadriannulatus). This result may have been due to a non-specific signal from one of the fluorescent probes or from DNA contamination during the setting up of the PCRs. The new assay also shows only a low level of ‘failed’ reactions in the trial with
The rapid identification of vector and non-vector species of the An. gambiae complex is often an important part of vector control strategies. Two high-throughput TaqMan assays have recently been developed for this purpose and both were shown to be significantly more sensitive than standard PCR (Bass et al., 2007; Walker et al., 2007). However, both assays have limitations. The Walker TaqMan assay is designed to detect and discriminate only An. gambiae s.s. and An. arabiensis and is unsuitable for testing other members of the complex (the assay incorrectly identifies An. quadriannulatus, An. merus and An. melas as An. gambiae s.s.). The Bass TaqMan assay distinguishes members of the complex in two groups, the main malaria vectors An. gambiae s.s. and An. arabiensis as one group and An. quadriannulatus, An. melas and An. merus as a second group. Used alone, the Bass TaqMan assay is unable to discriminate between An. gambiae s.s. and An. arabiensis which may be significant for certain studies, such as those examining the spread of resistance genes in mosquito populations. To overcome this, the two TaqMan assays can be used sequentially to further identify samples that are scored as An. gambiae s.s./An. arabiensis in the Bass TaqMan by subsequent testing using the Walker TaqMan. However, this has the obvious disadvantage of both increasing costs and lowering throughput. The new multiplex real-time PCR assay described here overcomes this by using three probes labelled with fluorophores with distinct emission and excitation spectra allowing sensitive and specific detection of (1) An. gambiae s.s. (2) An. arabiensis and (3) An. quadriannulatus/An. melas/An. merus as a group. Unfortunately, extensive examination of the available rRNA gene sequences did not reveal a suitable position to site additional probes that would allow the discrimination of An. quadriannulatus from An. melas and An. merus by this method. However, although An. melas and An. merus have been shown to be malaria vectors their distribution is limited to costal regions. In contrast, An. quadriannulatus species A is a nonvector species and is widespread in southern Africa where it is often
Table 1 Comparison of the genotyping results of the new multiplex PCR assay with data obtained previously by three alternative methods (Bass et al., 2007) Genotyping results
Correct scores Failed reactions Failed reactions excluding dilution series Incorrect scores
Bass TaqMan
AS-PCR
Walker TaqMan
Multiplex assay
455/466 11 5 0
364/466 99 60 3
333/371 25 11 2
421/466 31 12 2
C. Bass et al. / Acta Tropica 107 (2008) 50–53
sympatric with An. gambiae s.s. and/or An. arabiensis. Thus the new multiplex assay has application as (i) a vector/non-vector species identification test for large parts of sub-Saharan Africa (except for coastal regions) (ii) a means of distinguishing An. melas from An. gambiae s.s./An. arabiensis where they are sympatric such as regions along the west coast of Africa. The latter is possible because An. melas does not occur sympatrically with An. merus or An. quadriannulatus (Coetzee et al., 2000). It should, however, be used with caution in regions close to the east coast of Africa where An. quadriannulatus has been found to occur sympatrically with An. merus (Coetzee et al., 2000). Like the Bass TaqMan assay the new method will also correctly identify a ‘failed’ reaction as all members of the complex (except for An. bwambae which was not tested due to its rarity) are detected by one of the three fluorescent-labelled probes. The cost of the new multiplex PCR method is in the region of US $ 1 per mosquito analysed which represents a significant reduction in the cost of running the Bass and Walker TaqMan assays sequentially which costs in the region of US $ 1.5. The results of the genotyping trial carried out in this study showed that multiplexing the realtime PCRs did not result in any loss of sensitivity or specificity with 97% of samples correctly scored (compared to 96.5% and 98.75% for the Walker and Bass TaqMan assays using the same sample set). In addition, mosquitoes can be assigned to a species using a single leg without the need to first extract DNA, which increases throughput and allows further diagnostics (such as detection of the malaria parasite) to be conducted on the remaining tissue if required. Acknowledgements We thank Craig Wilding and Martin Donnelly (Liverpool School of Tropical Medicine) for collecting and organising the original mosquito samples. This work was supported by the Innovative Vector Control Consortium. Rothamsted Research receives grant-aided
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