2018 ASF Standard Operation Procedure 아프리카돼지열병진단개요 : - African swine fever, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Chapter2.

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1 2018 ASF Standard Operation Procedure 아프리카돼지열병진단개요 : - African swine fever, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Chapter OIE 2012 ver. - EU ASF reference lab CISA-INIA(Spain) diagnosis SOP ( ) Biosafety level - 3 (ABSL-3) (, ) - (PCR), (ELISA, IPT ) 2-2, 3, 2018 아프리카돼지열병 SOP_ 진단교육 ~9.13 1

2 아프리카돼지열병진단절차 2018 아프리카돼지열병 SOP_ 진단교육 ~9.13 2

3 샘플준비 1. ASF : (sera) ASF : EDTA,, (tissue homogenates), (soft tick s homogenates) : Gentamicin sulphate (50 mg / ml ), PBS 2. 1) - (jugular vein, inferior vena cava, articular vein ) 500 μl - : IPT, IFA - : IPT, IFA - (corporal fluids) : IPT, IFA - : 1 ml - : 1 ml - ( ) :,,,,,, 5g 2) (Soft tick s) Ornithodoros soft ticks carbon dioxide trapping, vacuum aspiration liquid nitrogen 3. 1) incubation rpm (780g) 10 3 ( ) 1.5 ml Eppendorf tube ( ) 아프리카돼지열병 SOP_ 진단교육 ~9.13 3

4 2) 2-1) incubation rpm (780g) 10 3 ( ) 1.5 ml Eppendorf tube 0.1% (Gentamicin) 4 1, tube ) 1 - : PBS 10 ( 1g 10 ml ) - (TissueLyser II): 50 mg + PBS 950 μl (1% )+steel ball 30Hz 4 2 3,000rpm(1,100g) 10 3 MINISTAR 0.45μl filter filtration 4 0.1% (Gentamicin) 4 1, tube 70, PCR ) 1 grinder 1.5ml cold PBS(0.1% gentamicin sulphate) ( automatic machine homogenize) 2 5,000g 5 3 tube 아프리카돼지열병 SOP_ 진단교육 ~9.13 4

5 DNA 추출 (Classical swine fever) total nucleic acid RNA (, DNase ) ASF DNA 1. High Pure PCR Template Preparation Kit (Manual) 1) : ASF nucleic acid : High Pure PCR Template Preparation Kit (Roche, cat no , 100 rxn) - ASFV DNA CSFV RNA multiplex PCR(Aguero et al., 2004) :,, homogenates,, homogenates 2) ul binding buffer 40 ul proteinase K (20mg/ml) 1.5 ml tube ul mix 72±2, 10 incubation ( volume 200 ul PBS 200 ul ) 3 spin down tube ul isopropanol 5 High Pure filter tube collection tube filter tube 6 8,000 rpm, 1 centrifuge ( centrifuge ) 7 collection tube clean collection tube filter tube ul Inhibitor Removal Buffer 8,000 rpm, 1 centrifuge 9 collection tube collection tube filter tube ul wash buffer 8,000 rpm, 1 centrifuge 11 clean collection tube 10 (washing) 12 clean collection tube 13,000 rpm, 10 centrifuge wash buffer ml tube filter tube ul (72±2 ) (CSFV RNA kit 2018 아프리카돼지열병 SOP_ 진단교육 ~9.13 5

6 elution buffer ) 8,000 rpm, 1 centrifuge * 200 μl elution buffer elution CISA-INIA SOP CSFV ASFV elution 15 DNA 10 (expiry date: 1 ) 1-2 PCR 4±3 3) Ct value 아프리카돼지열병 SOP_ 진단교육 ~9.13 6

7 항원진단 1. 1) : ASF p72 (Pirbright, UK) 2) Conventional PCR Bioneer AccuPower Premix/ Enzynomics 2X TOPsimple TM DyeMIN-nTaq PCR premix Eppendorf DE/Mastercycler pro 3) Real time PCR Bio-Rad iq supermix kit(biorad cat no 500rxn ) Bio-Rad CFX Connected / Bio-Rad CFX96 Touch 2. Conventional PCR Aguero et al., 2003 ASFV reference strain BA71V(Genbank ASU18466) VP72 target (88,363~88,619) real time PCR Forward primer 3 1nt mismatch 1) PCR primer Primer Sequence Size PPA-1 5'-AGTTATGGGAAACCCGACCC-3' 257bp PPA-2 5'-CCCTGAATCGGAGCATCCT-3' 2) Reaction mix : master mix Nuclease Free water PPA-1 (10pmol/ μl ) PPA-2 (10pmol/ μl ) Template DNA Total 17μl 0.5μl 0.5μl 2μl 20μl 3) PCR amplification : PCR Step Temperature/Time Cycle initial activation 95 10min 1cycle PCR denaturation annealing extension 95 15sec 62 30sec 72 30sec 40cycle Final extension 72 7min 1cycle 2018 아프리카돼지열병 SOP_ 진단교육 ~9.13 7

4) electrophoresis result Target band: 257 bp Enzynomics 2X TOPsimple TM DyeMIN-nTaq (primer 각 0.4 μl, sample DNA 3 μl적용 test 결과 ) 3. TaqMan R PCR (Real-time PCR) King et al.

8 4) electrophoresis result Target band: 257 bp Enzynomics 2X TOPsimple TM DyeMIN-nTaq (primer 각 0.4 μl, sample DNA 3 μl적용 test 결과 ) 3. TaqMan R PCR (Real-time PCR) King et al., 2003 ASFV reference strain BA71V(Genbank ASU18466) VP72 target QIAamp Viral Mini Kit (Qiagen, cat no 52904, 50rxn) : Swine fever 1) PCR primer Primer Primer 1 Primer 2 Probe Sequence 5'-CTGCTCATGGTATCAATCTTATCGA-3' (positive strand) 5'-GATACCACAAGATC(AG)GCCGT-3' (negative strand) FAM 5'-CCACGGGAGGAATACCAACCCAGTG- 3' TAMRA 2) Reaction mix : master mix Bio-Rad iq supermix kit Master mix reagents Biorad iq supermix(2x) Nuclease free water Primer 1 (50pM) Primer 2 (50pM) Probe (5pM) DNA template Total master mix volume 1x volume 12.5μl 6.5μl 1μl 1μl 1μl 3μl 25μl 3-1) PCR amplification: OIE manual (Fernandez-Pinero et al., 2010) 2018 아프리카돼지열병 SOP_ 진단교육 ~9.13 8

9 PCR Step Temperature/Time Cycle Polymerase activation 50 2min 1cycle DNA denaturation 95 10min 1cycle PCR amplification 95 15sec annealing 58 1min 40cycle 3-2) PCR amplification: CISA-INIA SOP/PCR/2 PCR Step Temperature/Time Cycle Activation of Taq 95 3min 1cycle DNA denaturation 95 10sec Primer annealing/elongation 58 30sec 45cycle 4-1) PCR amplification: OIE manual result Ct value >40.0, Ct value <40.0 ( Ct value <30.0) DNA copy number standard curve Ct value >38 sigmoidal plot doubtful, linear shape DNA copy number /ul ASF ) PCR amplification: CISA-INIA SOP/PCR/2 result E+(ASFV positive extraction control), R+(ASFV positive DNA reaction control) Ct value 32±4 E-(ASFV negative extraction control), R-(ASFV negative DNA reaction control) Ct value >40.0 positive sample Ct value <40.0, negative sample Ct value not report Ct value >38 sigmoidal plot doubtful, linear shape 2018 아프리카돼지열병 SOP_ 진단교육 ~9.13 9

10 4. Real-time PCR using universal probe lirary (UPL PCR, Fernndez-Pinero et al., 2013) CISA-INIA Universal Probe Library (UPL, Roche) 165presynthesized fluorogenic hydrolysis locked nucleic acid(lna) probe 8-9LNA residues in length, labeled with FAM dye (UPL#162, Roche) reference strain ASFV Spain70(Genebank S89966) VP bp, p72 genotype 1) PCR primer Primer Sequence ASF-VP72F 5'-CCCAGGRGATAAAATGACTG-3' ASF-VP72R 5'-CACTRGTTCCCTCCACCGATA-3' UPL162Probe(10pm/ μl ) 5'-6FAM -GGCCAGGA-dark quencher- 3' 2) Reaction mix : master mix LightCycler 480 Probes Master Kit (Roche, , 500rxn) Master mix reagents H 2 O Master mix 2X ASF-VP72F (20uM) ASF-VP72R (20uM) UPL 162probe 10uM RNA template Total master mix volume 1x volume 7μl 10μl 0.4μl 0.4μl 0.2μl 2μl 20μl 3) PCR amplification PCR Step Temperature/Time Cycle Activation of DNA polymerase 95 5min 1cycle DNA denaturation 95 10sec Primer annealing/elongation 60 30sec 45cycle 4) result E+(ASFV positive extraction control), R+(ASFV positive DNA reaction control) Ct value 32±4 E-(ASFV negative extraction control), R-(ASFV negative DNA reaction 2018 아프리카돼지열병 SOP_ 진단교육 ~

11 control) Ct value >40.0 positive sample Ct value <35.0, negative sample Ct value not report(ct value 40) Ct value 35 sigmoidal plot weak sample, 2 PCR duplicate 1 Ct value < 아프리카돼지열병 SOP_ 진단교육 ~

12 항체진단 1. ELISA (the prescribed test for international trade) : ELISA 1-1. Ingezim PPA compac kit (Ingenasa, Spain) 1) : INGEZIM PPA COMPAC (11.PPA.K3) -blocking ELISA -polystyrene plate VP72 major structural protein coating -mab conjugated with peroxidase - : ( ) 2) plate 4, control sera -20 washing solution(25x) : 40 ml w.s ml DW conjugate (100x) : 1plate conjugate 11 μl + 11 ml 3) 1 2 well diluent 50 μl G11,12 positive sera(pc) 50 μl, H11,12 negative sera(nc) 50 μl well 50 μl 2well <Plate layout> S1 S2 S3 S1 S2 S3 PC PC S40 S40 NC NC hr or overnight 4 NaOH plate 5 4 washing 6 conjugate 100 μl, washing 8 substrate(tmb) 100 μl, 15 9 stop solutioin 100 μl 2018 아프리카돼지열병 SOP_ 진단교육 ~

13 10 450nm reading 4) 1 negative control OD positive control OD 4 NC/PC>=4 2 positive cut off = NC-[(NC-PC)x0.5] negative cut off = NC-[(NC-PC)x0.4] PI=(NC-sample OD/NC-PC)*100 3 OD positive cut off, negative cut off 1-2. ID Screen African Swine Fever Indirect Screening (IDVET, France) IDVET ID Screen African Swine Fever Indirect Screening Test p32, p62, p72 recombinant protein plate Control 2 well, 1 well conjugate, control, substrate :,, meat juice, blood filter paper 1) preparation - DW wash solution 1x 2) test 1 - Dilution buffer 14 well 190 μl - Negative control A1, B1 10 μl - Positive control C1, D1 10 μl - well 10 μl min incubation 3 wash solution 300 μl 3 wash 4 dilution buffer 3 conjugate 10x 1x 5 conjugate 1x 100 μl well incubation 7 wash solution 300 μl 3 wash 8 substrate solution 100 μl well incubation in the dark 10 stop solution 100 μl well 11 OD 450nm reading 3) Result interpretation 2018 아프리카돼지열병 SOP_ 진단교육 ~

14 1 validation ODp C > OD PC /OD NC > 3 2 Interpretation S/P = (OD sample- OD NC / OD PC - OD NC ) 100 Result S/P 30% Status Negative 30% < S/P % 40% Doubtful S/N% 40% Positive 1-3. ID Screen African Swine Fever Indirect Confirmation IDVET ID Screen African Swine Fever Indirect - Biwell format p32, p62, p72 recombinant protein plate Control 4 well 2 well (No Ag strip, Ag strip ) conjugate, control, substrate screening test, 1) preparation - DW wash solution 1x 2) test 1 - Dilution buffer 14 well 190 μl - Negative control A1, A2, B1, B2 10 μl - Positive control C1, C2, D1, D2 10 μl - well 10 μl min incubation 3 wash solution 300 μl 3 wash 4 dilution buffer 3 conjugate 10x 1x 5 conjugate 1x 100 μl well incubation 7 wash solution 300 μl 3 wash 8 substrate solution 100 μl well incubation in the dark 10 stop solution 100 μl well 11 OD 450nm reading 3) Result interpretation 2018 아프리카돼지열병 SOP_ 진단교육 ~

1 validation ODp C > 0.350 OD PC /OD NC > 3 2 Interpretation S/P = (OD sample- OD NC / OD PC - OD NC ) 100 Result S/P 30% Status Negative 30% < S/P % 40% Doubtful S/N% 40% Positive 2.

15 1 validation ODp C > OD PC /OD NC > 3 2 Interpretation S/P = (OD sample- OD NC / OD PC - OD NC ) 100 Result S/P 30% Status Negative 30% < S/P % 40% Doubtful S/N% 40% Positive 2. Immunoperoxidase techinique : IPT (Confirmation test) 1),, dried blood filter paper IPT peroxidase enzyme vero MS cell, IPT alternative confirmatory test (EURL ) * ELISA IPT IPT plate EU reference lab CISA-INIA (ASF Coated 96-well plates fixed with ASFV adapted viruses(ba71v Vero or E70MS) belonging to p72 genotype) 2) 1 2 non infected cell < : CISA-INIA ASF SOP/IPT/1> 2018 아프리카돼지열병 SOP_ 진단교육 ~

16 Reference laboratories 1. OIE reference laboratories 2. EU FAO reference laboratory * ASF 2018 아프리카돼지열병 SOP_ 진단교육 ~

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PrimeScript 1st strand cdna Synthesis Kit 1 Central Dogma replication transcription 0 processing translation 오늘의내용은? 조직추출 RNAiso Plus 5 mrna 우리가관심있는 mrna가차지하는비율이극히일부이기때문에관심있는 mrna를증폭해서그것을눈으로확인할정도로만들어주는것이다.