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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

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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

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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

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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

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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

FLOW DIAGRAM

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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

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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

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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

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DCA # 26529MULTIPLEX HITS

1

3

4

6

13/19*

* SPECULATIVE HITS

HITS CONFIRMED WITH SINGLE SEROTYPING

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RESULT: DCA # 26529 CONTAINS BTV SEROTYPE 1

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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

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DCA # 26728MULTIPLEX A, B & C

A B C

26728

SINGLE SEROTYPING4 & 6

4 6

4/6

26728

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DCA # 26728MULTIPLEX HITS

4/6

* SPECULATIVE HITS

HITS CONFIRMED WITH SINGLE SEROTYPING

6

RESULT: DCA # 26728 CONTAINS BTV SEROTYPE 6

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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

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DCA # 26797MULTIPLEX HITS

3*

5*

7*

13/19

* SPECULATIVE HITS

HITS CONFIRMED WITH SINGLE SEROTYPING

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RESULT: DCA # 26797 CONTAINS BTV SEROTYPE 13

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SINGLE SEROTYPING3, 12 & 14

3

26801

12 14

DCA # 26801MULTIPLEX A, B & C

A B C

26801

12/14

3

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DCA # 26801MULTIPLEX HITS

3*

12/14

* SPECULATIVE HITS

HITS CONFIRMED WITH SINGLE SEROTYPING

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RESULT: DCA # 26801 CONTAINS BTV SEROTYPE 12

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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 &

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GEOGRAPHICAL DISTRIBUTION OF BTV SEROTYPES IDENTIFIED