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Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

Perera OP, Allen KC, Jain D, Purcell M, Little NS, Luttrell RG - J. Insect Sci. (2015)

Bottom Line: Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species.In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified.Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS Southern Insect Management Research Unit, Stoneville, MS 38776 op.perera@ars.usda.gov.

No MeSH data available.


Related in: MedlinePlus

Derivative dissociation curves and difference plots produced by the control and species-specific amplicons produced using optimized primer combination and squish buffer lysates from legs of H. armigera, H. zea, and 1:24 ratio of H. armigera: H. zea legs. KAPA YBR Fast Master Mix was used for DNA amplification. (A) Dissociation curves of H. armigera (red) and H. zea (blue) have the peaks from control amplicon and the species-specific amplicon. Mixed DNA (green) has all three peaks. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.
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iev137-F4: Derivative dissociation curves and difference plots produced by the control and species-specific amplicons produced using optimized primer combination and squish buffer lysates from legs of H. armigera, H. zea, and 1:24 ratio of H. armigera: H. zea legs. KAPA YBR Fast Master Mix was used for DNA amplification. (A) Dissociation curves of H. armigera (red) and H. zea (blue) have the peaks from control amplicon and the species-specific amplicon. Mixed DNA (green) has all three peaks. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.

Mentions: When HRM analysis was performed on the DNA amplified with optimal concentrations identified for 18S rRNA control primers and the species-specific ITS1 primers, only the peak representing the control amplicon (18S rRNA subunit) was produced in the reactions with DNA from Helio. subflexa and Helio. virescens (Fig. 3a). DNA from H. armigera and H. zea yielded peaks representing both the control amplicon and the amplicon specific to each species (Fig. 3a). The difference plot produced by HRM v2.0 software with H. zea as reference (Fig. 3b) produced distinct plots for H. armigera and H. zea that distinguished them from nontarget species Helio. subflexa and Helio. virescens (Fig. 3b). In addition to the species-specific dissociation curves produced by H. armigera and H. zea, DNA from artificial hybrids (mixture of DNA from both species) produced dissociation curves containing three peaks (Fig. 4a). These three peaks represented melting points of the 18S rRNA control amplicon and the species-specific amplicons of H. armigera and H. zea. The difference plots generated using the dissociation curves of each species and the hybrids were also readily distinguishable from each other (Fig. 4b). It was noted that the height of the dissociation curve peaks were directly correlated to the length of the amplicon and the quantity of amplified product. This effect is due to proportional variation in the strength of fluorescence signal from SYTO-9 dye intercalated to DNA molecules as observed with a low peak height of the 147 bp H. armigera-specific ITS1 amplicon compared with the 334 bp H. zea-specific amplicon (Figs. 3a and 4a). When dissociation curves were analyzed using HRM software, distinct difference plots were generated for H. armigera, H. zea, and the F1 hybrid male (Figs. 3b and 4b), facilitating reliable, semiautomated identification of H. armigera, H. zea, and F1 hybrids.Fig. 4.


Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

Perera OP, Allen KC, Jain D, Purcell M, Little NS, Luttrell RG - J. Insect Sci. (2015)

Derivative dissociation curves and difference plots produced by the control and species-specific amplicons produced using optimized primer combination and squish buffer lysates from legs of H. armigera, H. zea, and 1:24 ratio of H. armigera: H. zea legs. KAPA YBR Fast Master Mix was used for DNA amplification. (A) Dissociation curves of H. armigera (red) and H. zea (blue) have the peaks from control amplicon and the species-specific amplicon. Mixed DNA (green) has all three peaks. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4625950&req=5

iev137-F4: Derivative dissociation curves and difference plots produced by the control and species-specific amplicons produced using optimized primer combination and squish buffer lysates from legs of H. armigera, H. zea, and 1:24 ratio of H. armigera: H. zea legs. KAPA YBR Fast Master Mix was used for DNA amplification. (A) Dissociation curves of H. armigera (red) and H. zea (blue) have the peaks from control amplicon and the species-specific amplicon. Mixed DNA (green) has all three peaks. (B) Difference plot of dissociation curves generated by HRM v2.0 software using H. zea as the standard.
Mentions: When HRM analysis was performed on the DNA amplified with optimal concentrations identified for 18S rRNA control primers and the species-specific ITS1 primers, only the peak representing the control amplicon (18S rRNA subunit) was produced in the reactions with DNA from Helio. subflexa and Helio. virescens (Fig. 3a). DNA from H. armigera and H. zea yielded peaks representing both the control amplicon and the amplicon specific to each species (Fig. 3a). The difference plot produced by HRM v2.0 software with H. zea as reference (Fig. 3b) produced distinct plots for H. armigera and H. zea that distinguished them from nontarget species Helio. subflexa and Helio. virescens (Fig. 3b). In addition to the species-specific dissociation curves produced by H. armigera and H. zea, DNA from artificial hybrids (mixture of DNA from both species) produced dissociation curves containing three peaks (Fig. 4a). These three peaks represented melting points of the 18S rRNA control amplicon and the species-specific amplicons of H. armigera and H. zea. The difference plots generated using the dissociation curves of each species and the hybrids were also readily distinguishable from each other (Fig. 4b). It was noted that the height of the dissociation curve peaks were directly correlated to the length of the amplicon and the quantity of amplified product. This effect is due to proportional variation in the strength of fluorescence signal from SYTO-9 dye intercalated to DNA molecules as observed with a low peak height of the 147 bp H. armigera-specific ITS1 amplicon compared with the 334 bp H. zea-specific amplicon (Figs. 3a and 4a). When dissociation curves were analyzed using HRM software, distinct difference plots were generated for H. armigera, H. zea, and the F1 hybrid male (Figs. 3b and 4b), facilitating reliable, semiautomated identification of H. armigera, H. zea, and F1 hybrids.Fig. 4.

Bottom Line: Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species.In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified.Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs.

View Article: PubMed Central - PubMed

Affiliation: USDA-ARS Southern Insect Management Research Unit, Stoneville, MS 38776 op.perera@ars.usda.gov.

No MeSH data available.


Related in: MedlinePlus