Chapter 4: BIOINFORMATIC ANALYSIS AND PRIMER DESIGN
4.3 Results
Figure 4.4: The dendrogram (TreeView) produced from the full-length sequences of Segment 10 of the African horse sickness virus genome. The bar represents 0.1 substitutions per site.
Three distinct groupings exist in the dendrogram in Figure 4.4 as shown by the red circles. Figure 4.5 displays the dendrogram achieved after the alignment of the 10-190 bp region of Segment 10. This was the only region that complied with the conditions of being a single primer pair across all nine serotypes. These groupings of the full-length Segment 10 (Figure 4.4) dendrogram and the 10-190 bp regions (Figure 4.5) coincide.
Figure 4.5: The dendrogram (TreeView) produced from the 10-190 bp region of Segment 10 of the African horse sickness virus genome amplified by the primer pair AHS Seg10 10-190Fwd and AHS Seg10 10-190Rev. The bar represents 0.1 substitutions per site.
0.1
AHSV3 AJ007304 AHSV3 AJ007303 AHSV3 D12479
AHSV7 U60190 AHSV7 AJ007306
AHSV8 AJ007307 AHSV8 U02713
AHSV1 U02711 AHSV1 AM883173 AHSV2 U59279 AHSV6 U60189
AHSV5 AJ007309 AHS4 Z48735
AHSV6 U26171 AHSV9 D12480 AHSV9 AJ007308
AHSV4 Z48734 AHSV4 AJ007305 AHSV4 U02712
0.1
AHSV3 D12479 AHSV3 AJ007303 AHSV3 AJ007304 AHSV7 U60190 AHSV7 AJ007306
AHSVref AM883173 AHSV1 U02711 AHSV8 AJ007307 AHSV2 AFU59279 AHSV8 U02713 AHSV6 U60189
AHSV6 U26171 AHSV4 U02712 AHSV4 AJ007305 AHSV4 Z48734 AHSV9 D12480 AHSV9 AJ007308
AHSV4 Z48735 AHSV5 AJ007309
Serotypes 1, 2 and 8 have an identical amplicon and will not be distinguishable using the 10-190 bp region of Segment 10. Divergence within serotype 3 is low, while Serotype 7 is closely related to serotype 3. Serotype 6 is segregated from the nodes of serotype 4 and serotype 9. Serotype 5 exists in a node with one of the serotype 4‟s.
Three distinct groupings are easily identified, as shown by the circles.
The conditions for the selection of primers for the proposed assay were not met for Segment 2. Two primer sites on Segment 10, however, amplify a 180 bp region from position 10 to 190 across the 23 known sequences of the nine serotypes (Figure 4.6).
AHSV3_AJ007304 GTTTAAATTATCCCTTGTCATGAGTCTAGCTACGATCGCCGAAAATTATATGATGCATAA AHSV3_D12479 GTTTAAATTATCCCTTGTCATGAGTCTAGCTACGATCGCCGAAAATTATATGATGCATAA AHSV3_AJ007303 GTTTAAATTATCCCTTGTCATGAGTCTAGCTACGATCGCCGAAAATTATATGATGCATAA AHSV7_U60190 GTTTAAATTATCCCTTGTCATGAGTCTAGCTACGATCGCCGAAAATTATATGATGCATAA AHSV7_AJ007306 GTTTAAATTATCCCTTGTCATGAGTCTAGCTACGATCGCCGAAAATTATATGATGCATAA AHSV4_U02712 GTTTAAATTATCCCTTGTCATGAATCTAGCTGCAATCGCCAAGAATTATAGTATGCATAA AHSV4_AJ007305 GTTTAAATTATCCCTTGTCATGAATCTAGCTGCAATCGCCAAGAATTATAGTATGCATAA AHSV4_Z48734 GTTTAAATTATCCCTTGTCATGAATCTAGCTGCAATCGCCAAGAATTATAGTATGCATAA AHSV4_Z48735 GTTTAAATTATCC-TTGTCATGAATCTAGCTACAATCGCCAAGAATTATAGCATGCATAA AHSV5_AJ007309 GTTTAAATTATCCCTTGTCATGAATCTAGCTGCAATCGCCAAGAATTATAGCATGCATAA AHSV9_D12480 GTTTAA-TTATCCCTTGTCATGAATCTAGCTGCAATCGCCGAAAATTATAGTATGCATAA AHSV9_AJ007308 GTTTAA-TTATCCCTTGTCATGAATCTAGCTGCAATCGCCGAAAATTATAGTATGCATAA AHSV6_U26171 GTTTAA-TTATCCCTTGTCATGAATCTAGCTGCAATCGCCAAGAATTATAGTATGCATAA AHSV6_U60189 --TTAAATTATCCCTTGTCATGAATCTAGCTGCAATCGCCAAGAACTACAGTATGCATAA AHSVref_AM883173 GTTTAA-TTATCCCTTGTCATGAATCTTGCTAGCATCTCCCAAAGCTATATGTCACATAA AHSV1_U02711 GTTTAAATTATCCCTTGTCATGAATCTTGCTAGCATCTCCCAAAGCTATATGTCACATAA AHSV8_AJ007307 GTTTAAATTATCCCTTGTCATGAATCTTGCTAGCATCTCCCAAAGCTATATGTCACATAA AHSV2_AFU59279 GTTTAAATTATCCCTTGTCATGAATCTTGCTAGCATCTCCCAAAGCTATATGTCACATAA AHSV8_U02713 GTTTAAATTATCCCTTGTCATGAATCTTGCTAGCATCTCCCAAAGCTATATGTCACATAA **** ****** ********* *** *** *** ** * * ** * *****
AHSV3_AJ007304 TGGAAATCAGAGAGCAATTGTACCGTATGTTCCACCCCCTTATGCGTATGCAAATGCTCC AHSV3_D12479 TGGAAATCAGAGAGCAATTGTACCGTATGTTCCACCCCCTTATGCGTATGCAAATGCTCC AHSV3_AJ007303 TGGAAATCAGAGAGCAATTGTACCGTATGTTCCACCCCCTTATGCGTATGCAAATGCTCC AHSV7_U60190 TGAAACTCAGAGAGCAATTGTACCGTATGTTCCACCCCCTTATGCGTATGCAAATGCTCC AHSV7_AJ007306 TGGAAATCAGAGAGCAATTGTGCCGTATGTTCCACCCCCTTATGCGTATGCAAATGCTCC AHSV4_U02712 TGGAGAGTCGGGGGCGATTGTCCCTTATGTGCCACCGCCATATAATTTCGCGAGCGCTCC AHSV4_AJ007305 TGGAGAGTCGGGGGCGATTGTCCCTTATGTGCCACCGCCATATAATTTCGCGAGCGCTCC AHSV4_Z48734 TGGAGAGTCGGGGGCGATCGTCCCTTATGTGCCACCACCATACAATGTTGCGAGTGCTCC AHSV4_Z48735 TGGAGAGTCGGGGGCGATCGTCCCTTATGTGCCACCACCATACAATTTCGCAAGTGCTCC AHSV5_AJ007309 TGGAGAGTCGGGGACGATCGTCCCTTATGTGCCACCACCATACAATTTCGCAAGTGCTCC AHSV9_D12480 TGGAGAGTCGGGGGCGATCGTCCCTTATGTGCCACCACCATACAATTTCGCAAGTGCTCC AHSV9_AJ007308 TGGAGAGTCGGGGGCGATCGTCCCTTATGTGCCACCACCATACAATTTCGCAAGTGCTCC AHSV6_U26171 TGGAGAGTCGGAGGCGATCGTCCCTTATGTGCCACCACCATACAATTTCGCAAGTGCTCC AHSV6_U60189 TGGAGAGCAAGAGGCGATCGTCCCATATGTGCCGCCACCATACAATTTCGCAAGTGCTCC AHSVref_AM883173 TGAGAATGAAAGATCAATTGTACCATACATTCCGCCACCGTATCATC---CGACGGCTCC AHSV1_U02711 TGAGAATGAAAGATCAATTGTACCATACATTCCGCCACCGTATCATC---CGACGGCTCC AHSV8_AJ007307 TGAGAATGAAAGATCAATTGTACCATACATTCCGCCACCGTATCATC---CGACGGCTCC AHSV2_AFU59279 TGAGAATGAAAGATCAATTGTACCATACATTCCGCCACCGTATCATC---CGACGGCTCC AHSV8_U02713 TGAGAATGAAAGATCAATTGTACCATACATTCCGCCACCGTATCATC---CGACGGCTCC ** * ** ** ** ** * ** ** ** ** * * *****
Figure 4.6: The selection of two primer sites for the African horse sickness virus Segment 10 across the Clustal alignment of the 19 selected sequences located on GenBank. The primer sites are indicated in red and amplify a 180 bp product from position 10 to 190.
The primers, therefore, are as follows and amplify a 180 bp region from position 10-190 (Table 4.1):
Table 4.1: The selected primers and calculated properties to amplify a 180 bp product from position 10-190 of the African horse sickness virus Segment 10 (Bioneer, Daejeon, Korea)
Primer Sequence Length Tm
AHS Seg10 10-190Fwd ATCCCTTGTCATGARTCTWGCT 22 59.3°C AHS Seg10 10-190Rev CTTGACATKGCTTGRTTAAGTATCC 25 57.5°C
AHSV3_AJ007304 GACGCTTGGTGGTCAGGCGGGTGAAATGGAGTCCATGTCGCTTGGGATACTTAATCAAGC AHSV3_D12479 GACGCTTGGTGGTCAGGCGGGTGAAATGGAGTCCATGTCGCTTGGGATACTTAATCAAGC AHSV3_AJ007303 GACGCTTGGTGGTCAGGCGGGTGAAATGGAGTCCATGTCGCTTGGGATACTTAATCAAGC AHSV7_U60190 GACGCTTGGTGGTCAGGCGGGTGAAATGGAGTCCATGTCGCTTGGGATACTTAATCAAGC AHSV7_AJ007306 GACGCTTGGTGGTCAGGCGGGTGAAATGGAGTCCATGTCGCTTGGGATACTTAATCAAGC AHSV4_U02712 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV4_AJ007305 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV4_Z48734 GACGTTTTCTCAGGGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV4_Z48735 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV5_AJ007309 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV9_D12480 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV9_AJ007308 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV6_U26171 GACGTTTTCTCAGCGTACGAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAAGC AHSV6_U60189 GGCGCTTCCTCAGCGTACAAGTCAAATGGAGTCCGTGTCGCTTGGGATACTTAACCAGGC AHSVref_AM883173 GGCGCTTGCTGTATCCGCCAGTCAAATGGAGACCATGTCGCTTGGGATACTTAACCAAGC AHSV1_U02711 GGCGCTTGCTGTATCCGCCAGTCAAATGGAGACCATGTCGCTTGGGATACTTAACCAAGC AHSV8_AJ007307 GGCGCTTGCTGTATCCGCCAGTCAAATGGAGACCATGTCGCTTGGGATACTTAACCAAGC AHSV2_AFU59279 GGCGCTTGCTGTATCCGCCAGTCAAATGGAGACCATGTCGCTTGGGATACTTAACCAAGC AHSV8_U02713 GGCGCTTGCTGTATCCGCCAGTCAAATGGAGACCATGTCGCTTGGGATACTTAACCAAGC * ** ** * * ** ******** ** ******************* ** **
AHSV3_AJ007304 CATGTCAAGTACAACTGGTGCAAGTCGGGC---- AHSV3_D12479 CATGTCAAGTACAACTGGTGCAAGTCGGGC---- AHSV3_AJ007303 CATGTCAAGTACAACTGGTGCAAGTCGGGC---- AHSV7_U60190 CATGTCAAGTACAACTGGTGCAAGTGGGGC---- AHSV7_AJ007306 CATGTCAAGTACAACTGGTGCAAGTGGGGC---- AHSV4_U02712 CATGTCAAGTACAACTGGTGCGAGTGGGGC---- AHSV4_AJ007305 CATGTCAAGTACAACTGGTGCGAGTGGGGC---- AHSV4_Z48734 CATGTCAAATACAACTGGTGCGAGTGGGGC---- AHSV4_Z48735 CATGTCAAGTACAACTGGTGCGAGTGGGGCG--- AHSV5_AJ007309 CATGTCAAGTACAACTGGTGCGAGTGGGGC---- AHSV9_D12480 CATGTCAAGTACAACTGGTGCGAGTGGGGCG--- AHSV9_AJ007308 CATGTCAAGTACAACTGGTGCGAGTGGGGCG--- AHSV6_U26171 CATGTCAAGTACAACTGGTGCGAGTGGGGCG--- AHSV6_U60189 CATGTCAAGTACAACTGGTGCGAGTGGGGCAC-- AHSVref_AM883173 AATGTCAAGTTCAGCTGGTGCGAGCGGAGCACTT AHSV1_U02711 AATGTCAAGTTCAGCTGGTGCGAGCGGAGCACT- AHSV8_AJ007307 AATGTCAAGTTCAGCTGGTGCGAGTGGCGCACT- AHSV2_AFU59279 AATGTCAAGTTCAGCTGGTGCGAGTGGCGCACT- AHSV8_U02713 AATGTCAAGTTCAGCTGGTGCGAGTGGCGCACT- ******* * ** ******* ** * **
The annealing temperatures (Tm) of each primer were calculated using the OliCalc function in pDRAW32. Bioneer calculated the Tm‟s of the primer pair but did not provide any conditions or method of calculation for verification. The degeneracies of the primer pair require some minor adjustments to the primer pair in order to calculate an accurate Tm. The „bases‟ R, W and K may represent one of two bases – R: G/A; W: A/T and K:
G/T. Since A and T contribute to a lower melting point, they were substituted into the primer in order to calculate the Tm. The PCR reaction conditions also affect the Tm, and, as such, the following conditions were defined according to the Quantace SensiMix™ 2 step kit protocol: Primers = 200 nM; NaCl + KCl = 50 mM; MgCl2 = 3 mM; glycerol = 1
% (w/v); formamide = 0 % (w/v) and DMSO = 0 % (w/v).
The relative position of the primers on Segment 10 of the AHSV genome are shown in Figure 4.7 according to the program pDRAW32 (Tippmann, 2004).
AHSV Segment 10 Consensus Sequence (767 bp)
Figure 4.7: Relative positions of AHS Seg10 10-190Fwd (Fwd) and AHS Seg10 10-190Rev (Rev) with reference to a consensus sequence of 19 sequences of Segment 10 of the African horse sickness virus genome as defined by pDraw32. The numbers represent the position of each primer towards the centre of the amplicon.
Therefore, the 180 bp primer selection (AHS Seg10 10-190Fwd and AHS Seg10 10- 190Rev) is theoretically ensured of discriminating between the serotypes 9, 4 and 6; 3 and 7; and 1, 2 and 8. The significance of the primer selection in amplifying appropriate regions of the AHSV genome is that any field/vaccine strains of AHSV in serum will be identified to serotype.
A GenBank sequence representative of each serotype was used to determine the simulated melt profiles using POLAND (Steger, 1994 ) using the 10-190 bp region of Segment 10 (Figure 4.8).
AHSV Seg10 Consensus 767 bp
GC% in 1 bp blocks
0 10 20 30 40 50 60 70 80 90 100
==> Fwd - 31 <== Rev - 165
AHSV Seg10 Consensus 767 bp
GC% in 1 bp blocks
0 10 20 30 40 50 60 70 80 90 100
==> Fwd - 31 <== Rev - 165
AHSV1_U02711 AHSV2_AFU59279
AHSV3_D12479 AHSV4_AJ007305
AHSV5_AJ007309 AHSV6_U60189
Figure 4.8: Simulated melt profiles of the 10-190 bp regions of Segment 10 using POLAND. The serotype and GenBank accession number from which the amplicon was selected are found under each graph.
The red and blue lines use the predicted absorbance readings at 260 nm and 280 nm respectively to draw the graph and represent the change in absorbance over temperature.
AHSV7_AJ007306 AHSV8_AJ007307
AHSV9_D12480