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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.

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A. Gel phenotype of standard PCR for wild type An. gambiae s.l. from western Kenya, designated as hybrids by TaqMan genotyping. Lanes 1 – 11, field samples; Lane 12, a negative control. Lane 3 shows a true field hybrid with bands at the predicted 390 and 315 bp positions; lane 6 was a nonreactive sample in standard PCR. All other lanes are An. arabiensis by standard PCR, showing a predicted 315 bp product. B. Gel phenotype of standard PCR for wild type An. arabiensis and An. gambiae from western Kenya. Lanes 1 – 4, An. arabiensis by TaqMan genotyping. Lanes 5 – 9, An. gambiae by TaqMan genotyping. Lanes 10 – 12, undetermined by TaqMan genotyping, and standard PCR yielded no amplicon. A 390 bp amplicon is predicted for An. gambiae and a 315 bp amplicon is predicted for An. arabiensis. Lane M in both figures is a 1 Kb molecular weight ladder.
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Figure 3: A. Gel phenotype of standard PCR for wild type An. gambiae s.l. from western Kenya, designated as hybrids by TaqMan genotyping. Lanes 1 – 11, field samples; Lane 12, a negative control. Lane 3 shows a true field hybrid with bands at the predicted 390 and 315 bp positions; lane 6 was a nonreactive sample in standard PCR. All other lanes are An. arabiensis by standard PCR, showing a predicted 315 bp product. B. Gel phenotype of standard PCR for wild type An. arabiensis and An. gambiae from western Kenya. Lanes 1 – 4, An. arabiensis by TaqMan genotyping. Lanes 5 – 9, An. gambiae by TaqMan genotyping. Lanes 10 – 12, undetermined by TaqMan genotyping, and standard PCR yielded no amplicon. A 390 bp amplicon is predicted for An. gambiae and a 315 bp amplicon is predicted for An. arabiensis. Lane M in both figures is a 1 Kb molecular weight ladder.

Mentions: Because TaqMan genotyping experiments yielded 29 specimens classified as both An. gambiae and An. arabiensis, standard PCR was done on eleven of them, all females. Results showed that nine were An. arabiensis by gel phenotype, having a 315 bp amplicon, one sample did not react, and one sample had 315 and 360 bp amplicons, indicating a hybrid of both species or at least, both amplicons were present (Figure 3A). Additionally, the following samples were processed by standard PCR: four extractions classified as An. arabiensis, five classified as An. gambiae, and three classified as "undetermined" by TaqMan genotyping (Figure 3B). Of these, all of the successful classifications matched the standard PCR result, and further the 3 undetermined samples yielded no amplicons in standard PCR either.


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)

A. Gel phenotype of standard PCR for wild type An. gambiae s.l. from western Kenya, designated as hybrids by TaqMan genotyping. Lanes 1 – 11, field samples; Lane 12, a negative control. Lane 3 shows a true field hybrid with bands at the predicted 390 and 315 bp positions; lane 6 was a nonreactive sample in standard PCR. All other lanes are An. arabiensis by standard PCR, showing a predicted 315 bp product. B. Gel phenotype of standard PCR for wild type An. arabiensis and An. gambiae from western Kenya. Lanes 1 – 4, An. arabiensis by TaqMan genotyping. Lanes 5 – 9, An. gambiae by TaqMan genotyping. Lanes 10 – 12, undetermined by TaqMan genotyping, and standard PCR yielded no amplicon. A 390 bp amplicon is predicted for An. gambiae and a 315 bp amplicon is predicted for An. arabiensis. Lane M in both figures is a 1 Kb molecular weight ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: A. Gel phenotype of standard PCR for wild type An. gambiae s.l. from western Kenya, designated as hybrids by TaqMan genotyping. Lanes 1 – 11, field samples; Lane 12, a negative control. Lane 3 shows a true field hybrid with bands at the predicted 390 and 315 bp positions; lane 6 was a nonreactive sample in standard PCR. All other lanes are An. arabiensis by standard PCR, showing a predicted 315 bp product. B. Gel phenotype of standard PCR for wild type An. arabiensis and An. gambiae from western Kenya. Lanes 1 – 4, An. arabiensis by TaqMan genotyping. Lanes 5 – 9, An. gambiae by TaqMan genotyping. Lanes 10 – 12, undetermined by TaqMan genotyping, and standard PCR yielded no amplicon. A 390 bp amplicon is predicted for An. gambiae and a 315 bp amplicon is predicted for An. arabiensis. Lane M in both figures is a 1 Kb molecular weight ladder.
Mentions: Because TaqMan genotyping experiments yielded 29 specimens classified as both An. gambiae and An. arabiensis, standard PCR was done on eleven of them, all females. Results showed that nine were An. arabiensis by gel phenotype, having a 315 bp amplicon, one sample did not react, and one sample had 315 and 360 bp amplicons, indicating a hybrid of both species or at least, both amplicons were present (Figure 3A). Additionally, the following samples were processed by standard PCR: four extractions classified as An. arabiensis, five classified as An. gambiae, and three classified as "undetermined" by TaqMan genotyping (Figure 3B). Of these, all of the successful classifications matched the standard PCR result, and further the 3 undetermined samples yielded no amplicons in standard PCR either.

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