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MOLECULAR SEROTYPING OF BLUETONGUE VIRUS ISOLATES FROM THE 2014/2015 SEASON IN SOUTH
AFRICA K. GOOSEN; F. VAN DER MEER; I. RAUTENBACH; A. BOTHA; D GOOSEN*
*Corresponding Author
http://www.stackyard.com/news/images/fao/bluetongue.jpg
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PRIMER DESIGN •15 BTV
REFERENCE SEROTYPES
PCR OPTIMIZATIONBOTTLE
A
•BOTTLE A•SINGLE SEROTYPING PCR
•MULTIPLEX A BOTTLE A PCR
PCR OPTIMIZATION BOTTLE B AND
C
•BOTTLE B AND C
•SINGLE SEROTYPING PCR
•MULTIPLEX B
•MULTIPLEX C
FINAL PCR
CONDITIONS FOR
BOTTLE A,B,C
MULTIPLEX
PCR’S
•CONFIRM PCR CONDITIONS
•CONFIRM WORKING PRIMER CONCENTRATIONS
FLOW DIAGRAM
3
PRIMER DESIGN
https://upload.wikimedia.org/wikipedia/commons/thumb/9/91/Primers_RevComp.svg/2000px-Primers_RevComp.svg.png
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PRIMER DESIGN
• Based on 15 BTV reference serotypes found in Southern Africa
• OBP Vaccine (A,B & C) containing these 15 reference serotypes were used as template for molecular serotype developmentA - 1,4,6,12 and 14B - 3,8,9,10 and 11C – 2,5,7,13 and 19
• Primers designed to amplify as follow:Serotype 1 – 100 to 200 bpSerotype 2 – 200 to 300 bpSerotype 3 – 300 to 400 bpSerotype 4 – 400 to 500 bpSerotype 5 – 500 to 600 bpETC.
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PRIMER DESIGN
•15 BTV REFERENCE SEROTYPES
PCR OPTIMIZATIONBOTTLE
A
•BOTTLE A•SINGLE
SEROTYPING PCR
•MULTIPLEX BOTTLE A PCR
FLOW DIAGRAM
6
BOTTLE A SINGLE SEROTYPING PCRALL PRIMERS 0.4 µM
6 4
1412
1
95°C 95°C
55°C72°C 72°C
4°C
1 Min 15 sec
15 sec
10 sec 1 Min
35 Cycles
500 BP
1000 BP
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BOTTLE A SINGLE AND MULTIPLEX SEROTYPING PCR
DIFFERENTIAL PRIMER CONCENTRATIONS• 1- 0.2 µM• 4- 0.1 µM • 6- 0.2 µM • 12- 0.6 µM• 14- 0.6 µM
95°C 95°C
55°C72°C 72°C
4°C
1 Min 15 sec
15 sec
12 sec 1 Min
35 Cycles
8
BOTTLE A SINGLE AND MULTIPLEX SEROTYPING PCR
MULTIPLEX PRIMER CONCENTRATIONS
• 1- 0.2 µM• 4- 0.1 µM • 6- 0.2 µM • 12- 0.6 µM• 14- 0.6 µM
95°C 95°C
55°C72°C 72°C
4°C
1 Min 15 sec
15 sec
90sec 1 Min
35 Cycles
6 4
1412
1
9
PRIMER DESIGN •15 BTV REFERENCE SEROTYPES
PCR OPTIMIZATIONBOTTLE A
•BOTTLE A•SINGLE SEROTYPING PCR•MULTIPLEX BOTTLE A PCR
PCR OPTIMIZATION BOTTLE B AND C•BOTTLE B AND C•SINGLE SEROTYPING PCR•MULTIPLEX B•MULTIPLEX C
FLOW DIAGRAM
10
BOTTLE B AND C SINGLE SEROTYPING PCR ALL PRIMERS 0.4 µM
95°C 95°C
55°C72°C 72°C
4°C
1 Min 15 sec
15 sec
15 sec 1 Min
35 Cycles
3 8 9 10 11 2 5 7 13 19
11
BOTTLE B AND C SINGLE SEROTYPING PCR SEROTYPES 7, 8, 9, 10, 11, 13 & 19
ALL PRIMERS 0.6 µM
8 9 10 117 13 197 19
12
BOTTLE B AND C MULTIPLEX SEROTYPING PCR
all primers 0.6 µM
8 9 10 117 137 19 CB
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PRIMER DESIGN •15 BTV REFERENCE SEROTYPES
PCR OPTIMIZATIONBOTTLE A
•BOTTLE A•SINGLE SEROTYPING PCR•MULTIPLEX BOTTLE A PCR
PCR OPTIMIZATION BOTTLE B AND C
•BOTTLE B AND C•SINGLE SEROTYPING PCR•MULTIPLEX B•MULTIPLEX C
FINAL PCR CONDITIONS FOR BOTTLE A,B,C MULTIPLEX PCR’S•CONFIRM PCR CONDITIONS•CONFIRM WORKING PRIMER CONCENTRATIONS
FLOW DIAGRAM
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FINAL MULTIPLEX SEROTYPING PCR’S
95°C 95°C
55°C72°C 72°C
4°C
1 Min 15 sec
15 sec
90sec 1 Min
35 CyclesMULTIPLEX PRIMER CONCENTRATIONSBOTTLE A• 1- 0.2 µM• 4- 0.1 µM 6- 0.2 µM • 12- 0.6 µM• 14- 0.6 µm
MULTIPLEX PRIMER CONCENTRATIONSBOTTLE B• 3- 0.1 µM• 8- 0.5 µM • 9- 0.5 µM • 10- 0.6 µM• 11- 0.6 µM
MULTIPLEX PRIMER CONCENTRATIONSBOTTLE C• 2- 0.1 µM• 5- 0.1 µM • 7- 0.5 µM • 13- 0.6 µM• 19- 0.6 µM
A B C
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PRIMER DESIGN •15 BTV REFERENCE SEROTYPES
PCR OPTIMIZATIONBOTTLE A
•BOTTLE A•SINGLE SEROTYPING PCR•MULTIPLEX BOTTLE A PCR
PCR OPTIMIZATION BOTTLE B AND C•BOTTLE B AND C•SINGLE SEROTYPING PCR•MULTIPLEX B•MULTIPLEX C
FINAL PCR CONDITIONS FOR BOTTLE A,B,C MULTIPLEX PCR’S •CONFIRM PCR CONDITIONS•CONFIRM WORKING PRIMER CONCENTRATIONS
BTV SEROTYPING OF DCA FIELD ISOLATES 2014/2015 WITH THE OPTIMIZED PCR •FIRST A, B & C MULTIPLEX PCR•CONFIRM SUSPECTED HITS WITH SINGLE SEROTYPING PCR
FLOW DIAGRAM
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DCA # 26529MULTIPLEX A, B & C
A B C
26529
SINGLE SEROTYPING1, 3, 4, 6, 13 & 19
1 3 4 6 13
1,4,6
3
13/19
19
26529
17
DCA # 26529MULTIPLEX HITS
1
3
4
6
13/19*
* SPECULATIVE HITS
HITS CONFIRMED WITH SINGLE SEROTYPING
1
RESULT: DCA # 26529 CONTAINS BTV SEROTYPE 1
18
DCA # 26650MULTIPLEX A, B & C
A B C
26650
SINGLE SEROTYPING1, 3, 13 & 19
1 3 13
13
19
13/19
26650
19
DCA # 26650MULTIPLEX HITS
1
3*
13/19*
* SPECULATIVE HITS
HITS CONFIRMED WITH SINGLE SEROTYPING
1
RESULT: DCA # 26650 CONTAINS BTV SEROTYPE 1
20
DCA # 26728MULTIPLEX A, B & C
A B C
26728
SINGLE SEROTYPING4 & 6
4 6
4/6
26728
21
DCA # 26728MULTIPLEX HITS
4/6
* SPECULATIVE HITS
HITS CONFIRMED WITH SINGLE SEROTYPING
6
RESULT: DCA # 26728 CONTAINS BTV SEROTYPE 6
22
SINGLE SEROTYPING3, 5, 7, 13 & 19
3 5
26797
7 13 19
DCA # 26797MULTIPLEX A, B & C
A B C
26797
4/6
13/195,7
3
23
DCA # 26797MULTIPLEX HITS
3*
5*
7*
13/19
* SPECULATIVE HITS
HITS CONFIRMED WITH SINGLE SEROTYPING
13
RESULT: DCA # 26797 CONTAINS BTV SEROTYPE 13
24
SINGLE SEROTYPING3, 12 & 14
3
26801
12 14
DCA # 26801MULTIPLEX A, B & C
A B C
26801
12/14
3
25
DCA # 26801MULTIPLEX HITS
3*
12/14
* SPECULATIVE HITS
HITS CONFIRMED WITH SINGLE SEROTYPING
12
RESULT: DCA # 26801 CONTAINS BTV SEROTYPE 12
26
SINGLE SEROTYPING3, 4, 8, 9, 10 & 11
3
25394
10 11
DCA # 25394MULTIPLEX A, B & C
A B C
25394
4
4
4
8,9,10,113
4 8 9
438 9 10 11
A B C
POS ITIVE CONTROL
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DCA # 25394MULTIPLEX HITS
3
4
8/9/10/11
* SPECULATIVE HITS
HITS CONFIRMED WITH SINGLE SEROTYPING
3, 4, 8, 9, 10 & 11
RESULT: DCA # 25394 CONTAINS BTV SEROTYPES 3, 4, 8, 9, 10 & 11
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CONCLUSION
• MULTIPLEX PCR’S ARE MORE SENSITIVE THAN SINGLE SEROTYPING PCR’S
• ISOLATE CONSIDERED POSITIVEIF A SEROTYPE WAS IDENTIFIED BY BOTH MULTILEX AND SINGLE PCR
• MULTIPLEX PCR SCREENING DONE FIRST• HITS CONFIRMED WITH LESS SENSTIVE SINGLE SEROTYPING
PCR• SEROTYPE 11 WAS NOT IDENTIFIABLE IN THE POSITIVE
CONTROL, HOWEVER IT WAS FOUND IN ISOLATE 25394• FURTHER OPTIMIZATION AND SEQUENCING WILL BE
CONDUCTED ON ALL 26 KNOWN BLUETONGUE VIRUS SEROTYPES
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ISOLATE SEROTYPE(S)26529 126650 126728 626801 1226797 1325394 3, 4, 8, 9, 10 &
11
GEOGRAPHICAL DISTRIBUTION OF BTV SEROTYPES IDENTIFIED
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