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2019 KASV
2019 KASV
수의양돈정책포럼
수의양돈정책포럼
V E T T O G E T H E R
일시 2019년 3월 26일 [화]장소 충남대학교 정심화국제문회회관 백마홀
2019.3.26 www.KASV.or.kr
www.KASV.or.kr
2019KASV
수의양돈정책포럼
주최 주관 / 김현권 의원주최 주관 / 김현권 의원
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65구제역바이러스 현장진단킷트 개발 및 사용례
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구제역바이러스 현장진단키트 개발 및 사용례
㈜메디안디노스틱
2019. 03. 26
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1. 구제역바이러스 혈청형 구분 현장진단키트의 필요성
2. 구제역바이러스 현장진단키트의 개발 3. 구제역바이러스 현장진단키트의 민감도 / 특이도
4. 구제역바이러스 현장진단키트의 사용례
목차
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1. 구제역바이러스 구 현장진단키트
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Distribution of the seven endemic pools of Foot-and-Mouth Disease showing conjectured status of Foot-and-Mouth Disease in countries during 2015.
1. 구제역바이러스 구 현장진단키트
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Reference : OIE/FAO Foot-and-Mouth Disease Referene Laboratory Network Annual Report 2017
1. 구제역바이러스 구 현장진단키트
ELISA
PCR
PRRS 진단 및 현장진단 사(POCT)
이 개 사
monitoring
현장진단 사
(5~15min)
Culture
9 이 9 혈청형(7 ) 의 차
1. 구제역바이러스 구 현장진단키트
7 2016 1 1 ~ 2017 12 31 (2 )
2. 구제역바이러스 현장진단키트 개발- 역 구
구제역진단
Immunization of mouse
Cell fusion
Expansion and selection of cultures to be cloned
Isolation and expansion of clones
Antibody titration
Isolation of spleen cells Preparation of myeloma cells
Screening of hybridoma supernatants
Cloning
Production of ascitic fluids
Purification and Final testing of monoclonal antibodies
Monoclonal antibody production technology
2. 구제역바이러스 현장진단키트 개발 - FMDV 이 개발 scheme
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Colloidal gold Lateral Flow Immunochromatographic Assay
2. 구제역바이러스 현장진단키트 개발 – assay platform
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FMDV O, A, Asia, Pan 100 mAb 제 mAb matching test Æ setotype(O,A,Asia1) specific & Pan
2. 구제역바이러스 현장진단키트 개발 – mAb 제 및 이 mAb 발
11 11 11
Nitrocellulose
membrane
Absorption pad
Gold
conjugate pad
Sample pad
Control line
Test line
Mouse anti-Malaria rAg specific mAb
Mouse anti-FMDV A specific mAb [6E82] (Capture)
C
Mouse anti-FMDV (all serotypes) specific mAb
[1E58] (Detector) conjugated gold nanoparticle
A
Positive at T line
Capture mAb
Detector mAb
FMDV binding by
sandwich method
FMDV O, A, Asia1 serotypes
Mouse anti-FMDV Asia1 specific mAb [5D39] (Capture)
Mouse anti-FMDV O specific mAb [4B14] (Capture) O
AS
Malaria rAg conjugated gold nanoparticle
Strip1 3Diff
2. 구제역바이러스 현장진단키트 개발 – 진단키트 구 및
12 12 12
Nitrocellulose membrane
Absorption pad
Gold conjugate pad
Sample pad
Control line
Test line
Mouse anti-Malaria rAg specific mAb
Mouse anti-FMDV (all serotypes) specific mAb [1E58] (Capture)
C
PAN
Positive at T line Capture mAb
Detector mAb
FMDV binding by sandwich method
FMDV all serotypes (O, A, Asia1, C, SAT1, SAT2, SAT3)
Malaria rAg conjugated gold nanoparticle
Strip2 PAN
Mouse anti-FMDV (all serotypes) specific mAb [1E58] (Detector) conjugated gold nanoparticle
2. 구제역바이러스 현장진단키트 개발 – 진단키트 구 및
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C
C O A Asia1
C
C
C
As A O
1) Sample loading to the rapid kit for FMDV O Ag detection
2) Primary antigen-antibody reaction in gold conjugate pad
3) Secondary antigen-antibody reaction on test lines in nitrocellulose membrane
4) Unbound molecules(antigen or antibody) are moved to absorption pad
O A Asia1
O A Asia1
O A Asia1
2. 구제역바이러스 현장진단키트 개발 – 진단키트 구 및
14 FMDV serotype A positive
FMDV serotype O positive FMDV serotype Asia1 positive
FMDV other serotype (SAT1, SAT2, SAT3, C) positive
Negative
2. 구제역바이러스 현장진단키트 개발 – 제 및
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키트의 구성 성
사 사 요
2. 구제역바이러스 현장진단키트 개발 – 사
Yang-Ju Jincheon O/Hapcheon/KOR/2014
Gimje Boeun Jeongeup O1manisa O1/BFS/860/UK/67
Yunlin/Taiwan/97
ME-SA Euro-SA Cathay
Strip 3Diff 5.5 3.9 3.7 3.9 4.9 4.3 4.7 3.9 4.0
Strip PAN 4.5 3.9 3.7 3.9 4.9 4.3 3.7 3.9 4.0
5.5 3.9 4.7 3.9 >4.9 5.3 4.7 4.7 5.0
5.5 3.9 4.7 3.9 >4.9 5.3 4.7 4.7 5.0
MEDIAN
SVANOVA
PBM
Kit
Limit of Detection in FMDV O Strains (LogTCID50/ml)
SEA/Mya-98 ME-SA/ind-2001d
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¾ Limit of Detection is Same or Better (10 times) than PBM(USA), SVANOVA
� Limit of Detection in FMDV O strain (9 different topotypes)
104.7 103.7 102.7 104.7 103.7 103.7 102.7 104.7 103.7 102.7
MEDIAN SVNOVA PBM
Hapcheon + + + w+ - -
+ + - - - -
3. 구제역바이러스 현장진단키트 / 이 – Analytical sensitivity
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Pocheon Yeoncheon Malaysia97 P1A-189 Iran05 A22 Iraq
FMDVA/SAU/2/2015
Asia/Iran-05 Asia/G-IV
Strip 3Diff 5.4 4.9 4.0 4.4 4.5 4.7
Strip PAN 5.4 4.9 4.0 4.4 4.5 3.7
>5.4 >4.9 5.0 4.4 4.5 4.7
5.4 4.9 5.0 4.4 5.5 4.7
MEDIAN
SVANOVA
PBM
Asia/Sea-97
Kit
Limit of Detection in FMDV A Strains (LogTCID50/ml)
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¾ Limit of Detection is Same or Better (10 times) than PBM(USA), SVANOVA
� Limit of Detection in FMDV A strain (6 different topotypes)
105.0 104.0 103.0
MEDIAN SVNOVA PBM
Malaysia + + w+ w+ - -
+ + - - - -
103.0 103.0 104.0 104.0 105.0 105.0
3. 구제역바이러스 현장진단키트 / 이 – Analytical sensitivity
MOG/05 CAM/9/80 Sharmir
G-V
Strip 3Diff 4.9 4.6 4.7
Strip PAN 4.9 4.6 4.7
4.9 5.6 >4.7
4.9 5.6 4.7
MEDIAN
SVANOVA
PBM
Limit of Detection in FMDV Asia1 Strains (LogTCID50/ml)
Kit
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¾ Limit of Detection is Same or Better (10 times) than PBM(USA), SVANOVA
� Limit of Detection in FMDV Asia1 strain (3 different topotypes)
105.6 104.6 103.6
MEDIAN SVNOVA PBM
CAM/9/80 + + w+ w+ - -
+ + - - - -
103.6 103.6 104.6 104.6 105.6 105.6
3. 구제역바이러스 현장진단키트 / 이 – Analytical sensitivity
SAT1 SAT2 SAT3 C
SAT1/BOT/1/68 SAT2/ZIM/5/81 SAT3/ZIM/4/81 C3/Resende/BRA/55
WZ(Ⅲ) WZ(Ⅱ) Euro-SA
Strip 3Diff - - - -
Strip PAN 5.6 4.9 4.6 3.7
5.6 >4.9 5.6 4.7
>5.6 >4.9 >5.6 4.7
SVANOVA
PBM
Limit of Detection in FMDV (LogTCID50/ml)
Kit
MEDIAN
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¾ Limit of Detection is Same or Better (10 times) than PBM(USA), SVANOVA
� Limit of Detection in FMDV SAT1, SAT2, SAT3, C strain
105.7 104.7 103.7
MEDIAN SVNOVA PBM
C3 + w+ - -
+ + - - - -
103.7 103.7 104.7 104.7 105.7 105.7
- -
3. 구제역바이러스 현장진단키트 / 이 – Analytical sensitivity
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¾ No Cross-reaction with other viruses(5) including vesicular disease associated viruses
� Cross reactivity test (other vesicular disease associated viruses)
Vesicular stomatitis virus Negative Negative
Swine vesicular disease virus Negative Negative
Seneca valley virus Negative Negative
Porcine reproductive and respiratory syndrome virus Negative Negative
Classical swine fever virus Negative Negative
Virus Strip 3Diff Strip PAN
3. 구제역바이러스 현장진단키트 / 이 – Analytical specificity
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¾ No Cross-reaction with other viruses(5) including vesicular disease associated viruses
� Cross reactivity test (Serotypes)
Vesicular stomatitis virus Negative Negative
Swine vesicular disease virus Negative Negative
Seneca valley virus Negative Negative
Porcine reproductive and respiratory syndrome virus Negative Negative
Classical swine fever virus Negative Negative
Virus Strip 3Diff Strip PAN
3. 구제역바이러스 현장진단키트 / 이 – Analytical specificity
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¾ No Cross reaction between different serotypes
� Culture supernatants containing different type of FMDV (A, Asia, C, SAT1, 2, 3)
Strip PAN
O A Asia 1 PAN
O(9) Positive Negative Negative Positive
A(6) Negative Positive Negative Positive
Asia 1(3) Negative Negative Positive Positive
SAT 1(1) Negative Negative Negative Positive
SAT 2(1) Negative Negative Negative Positive
SAT 3(1) Negative Negative Negative Positive
C(1) Negative Negative Negative Positive
Type(No. of strains)
Results
Strip 3Diff
3. 구제역바이러스 현장진단키트 / 이 – Analytical specificity
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¾ Higher Sensitivity than PBM, SVANOVA in every serotypes
� Clinical Sensitivity
1) Test samples used in this test were already determined as positive by PCR or ELISA 2) No serotype-cross reaction between test line
MEDIAN2) PBM SVANOVA
Saliva 70% (13/18) 0% (0/13) 67% (6/9)Vesicular fluid 100% (3/3) 100% (1/1) NT
Tissue 94% (44/47) 86% (25/29) NTTotal 88% (60/68) 60% (26/43) NT
AVirus-Spiked Saliva
(=Total)100% (70/70) 10% (5/25) 40% (8/20)
Tissue 100% (1/1) 100% (1/1) NTVirus-Spiked Saliva 100% (60/60) 52% (31/60) 50% (6/12)
Total 100% (61/61) 52% (32/61) 50% (6/12)
SAT1 100% (20/20) NT 0% (0/4)
SAT2 100% (20/20) NT 0% (0/4)
SAT3 100% (20/20) NT 0% (0/4)
C 100% (20/20) 75% (15/20) 100% (4/4)
Sensitivity1)
(No. of Positive/No. of Samples)
O
Serotype Type of Samples
Asia1
Virus-Spiked Saliva(=Total)
3. 구제역바이러스 현장진단키트 / 이 – Clinical sensitivity
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¾ FMDV negative Saliva in Korea (RT-PCR)
9 Specificity in bovine: 100% (n=92/92)
9 Specificity in swine: 99.5% (n= 398/400)
� Total 99.6% specificity (n= 490/492)
� Clinical Specificity
3. 구제역바이러스 현장진단키트 / 이 – Clinical specificity
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구
17/07/07 10-2017-0086716 구제역바이러스 혈청형 O 용 단 이의 용도 민 ( 역 )
스
17/10/13 10-2017-0133574 구제역 바이러스 혈청형 A 용 단 이의 용도 민 ( 역 )스
17/11/21 10-2017-0155390 구제역 바이러스 혈청형 Asia1 용 단 이의 용도 민 ( 역 )
스
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및
27
2017 Global FMD Research Alliance, p63, 2017
발
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Date Country Estimated lesion age (days)
Lesion site
Sample type
Svanova Median (pan)
Median (serotype)
Lab result
Note
21/11/2017 Kenya 1-2 Oral Epithelium - - - SAT2 Svanova buffer used in median kits
21/11/2017 Kenya 1-2 Oral Epithelium + (weak) - - SAT2 Svanova buffer used in median kits
28/11/2017 Kenya 7-10 Foot Epithelium - + (weak) - SAT2 Svanova buffer used in median kits
28/11/2017 Kenya 4-5 Oral Epithelium - - - SAT2 Svanova buffer used in median kits
27/02/2018 Nepal 2-3 Oral Epithelium - + + (O) O
13/03/2018 Nepal 1-2 Foot Epithelium Not recorded
+ + (O) O
13/03/2018 Nepal 1-2 Foot Epithelium Not recorded
+ + (O) O
20/03/2018 Nepal 2 Foot Epithelium Not recorded
- - O
20/03/2018 Nepal 2 Foot Epithelium Not recorded
+ + (O) O
20/03/2018 Nepal 2 Oral Epithelium Not recorded
+ + (O) O
20/03/2018 Nepal 1-2 Foot Epithelium Not recorded
+ + (O) O
20/03/2018 Nepal 2 Oral Epithelium Not + Not recorded O
FAO (2017. 11. 21 ~ 2018. 03. 20)
4. 구제역바이러스 현장진단키트 사용례
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FAO (2017. 11. 27 ~ 2018. 12. 04) 4. 구제역바이러스 현장진단키트 사용례
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FAO (2017. 11. 27 ~ 2018. 12. 04) 4. 구제역바이러스 현장진단키트 사용례
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Mongolia - SCVL (2018. 03. 30) 4. 구제역바이러스 현장진단키트 사용례
type A (epitelium) A/Asia/Sea-97-2016
type Asia1 (epithelium)-2005
State Central Veterinary Laboratory(SCVL) in Mongolia
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사 .