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Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

Walker ED, Thibault AR, Thelen AP, Bullard BA, Huang J, Odiere MR, Bayoh NM, Wilkins EE, Vulule JM - Malar. J. (2007)

Bottom Line: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR.In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. walker@msu.edu

ABSTRACT

Background: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan single nucleotide polymorphism (SNP) genotyping.

Methods: Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates.

Results: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.

Conclusion: TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

Show MeSH
Bivariate plot generated from TaqMan genotyping, given as output from the ABI Prism 7900HT Sequence Detection System (proprietary hardware and software from Applied Biosystems). Samples are from wild caught An. gambiae s.l. adults from western Kenya. Red dots are An. gambiae s.s., blue dots are An. arabiensis, green dots are hybrids (two samples) or plasmid controls (three samples), black x's are undetermined samples, and black squares are internal negative controls.
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Figure 2: Bivariate plot generated from TaqMan genotyping, given as output from the ABI Prism 7900HT Sequence Detection System (proprietary hardware and software from Applied Biosystems). Samples are from wild caught An. gambiae s.l. adults from western Kenya. Red dots are An. gambiae s.s., blue dots are An. arabiensis, green dots are hybrids (two samples) or plasmid controls (three samples), black x's are undetermined samples, and black squares are internal negative controls.

Mentions: Of 3,041 field caught An. gambiae s.l. analysed by TaqMan genotyping, there were 2,621 successful classifications, 391 undetermined specimens, and 29 specimens classified as both species, giving a success rate of 87.02% when counting the classifications of "both" as a negative result. There were 1,223 males and 1,398 females amongst the successful reactions, of which 804 males and 766 females were An. gambiae s.s., and the remainder were An. arabiensis. Overall, the composition of the community was 51.63% An. gambiae s.s., and 48.37% An. arabiensis. Output as a bivariate plot from the classification system is shown in Figure 2.


Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping.

Walker ED, Thibault AR, Thelen AP, Bullard BA, Huang J, Odiere MR, Bayoh NM, Wilkins EE, Vulule JM - Malar. J. (2007)

Bivariate plot generated from TaqMan genotyping, given as output from the ABI Prism 7900HT Sequence Detection System (proprietary hardware and software from Applied Biosystems). Samples are from wild caught An. gambiae s.l. adults from western Kenya. Red dots are An. gambiae s.s., blue dots are An. arabiensis, green dots are hybrids (two samples) or plasmid controls (three samples), black x's are undetermined samples, and black squares are internal negative controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808465&req=5

Figure 2: Bivariate plot generated from TaqMan genotyping, given as output from the ABI Prism 7900HT Sequence Detection System (proprietary hardware and software from Applied Biosystems). Samples are from wild caught An. gambiae s.l. adults from western Kenya. Red dots are An. gambiae s.s., blue dots are An. arabiensis, green dots are hybrids (two samples) or plasmid controls (three samples), black x's are undetermined samples, and black squares are internal negative controls.
Mentions: Of 3,041 field caught An. gambiae s.l. analysed by TaqMan genotyping, there were 2,621 successful classifications, 391 undetermined specimens, and 29 specimens classified as both species, giving a success rate of 87.02% when counting the classifications of "both" as a negative result. There were 1,223 males and 1,398 females amongst the successful reactions, of which 804 males and 766 females were An. gambiae s.s., and the remainder were An. arabiensis. Overall, the composition of the community was 51.63% An. gambiae s.s., and 48.37% An. arabiensis. Output as a bivariate plot from the classification system is shown in Figure 2.

Bottom Line: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR.In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. walker@msu.edu

ABSTRACT

Background: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan single nucleotide polymorphism (SNP) genotyping.

Methods: Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates.

Results: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.

Conclusion: TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

Show MeSH