PT CENTRAL PROTEINA PRIMA, Tbk.

ISOLASI DNA Vp AHPND DARI

MEDIA CAIR DAN ORGAN

Disampaikan pada acara Bimbingan Teknis Deteksi AHPND pada Komoditi Udang, 3 Juli 2019
ENRICHMENT SAMPEL UJI AHPND
 Preservasi sample AHPND

Studi dilakukan pada 2 jenis udang, yaitu L vanammei dan

P. monodon.
 Hasil Analisa

Untuk O1, P1 dan Q1 positive berkisar pada 3.3% - 16.7%)

Sedangkan O2, P2 dan Q2 berkisar pada 25 – 100% (Vpldh),
12.5 – 100% untuk VpPirAB dan 37.5 – 100% untuk AP4.
MANFAAT ENRICHMENT
MANFAAT ENRICHMENT
• meningkatkan positivity
sampel / potensi
terdeteksinya sampel positif :
~ mencegah FALSE NEGATIVE
• Mempertebal band hasil
elektroforesis (Han et al,
2017)

Apakah akan mengubah

sampel yang negatif menjadi
positif?
PERBANDINGAN HASIL ENRICHMENT SAMPEL

Catatan:
- Tanpa enrichment,
semua sampel
akan negatif
- Bila sampel
negatif,
setelah enrichment
akan tetap negatif
- Minimum 18 jam
- Perlu surveillance
ISOLASI AHPND DARI SAMPLE AIR
dan LUMPUR
Kertas saring masukkan
ke dalam 2 ml TSB

100 ml sample
Inkubasi pada room
temp selama 18 jam

Whatman Glass Microfiber

Filter GF/CTM diameter 47
mm Cat. No. 1822-047
 ISOLASI AHPND DARI ORGAN di
HATCHERY
• Ambil sampel nauplii/larva/PL/artemia/cumi/polychaeta, sebanyak 1 gr,
masukkan ke dalam tabung reaksi ukuran 10 mL (PL 5 : 500 ekor; PL 7: 400
ekor; PL 10: 150 ekor)
• Tambahkan 500 µL larutan NaCl 0.85%, gerus sampai hancur dengan
batang penggerus atau vorteks hingga tercampur merata.
• Homogenase larutan sampel tersebut dengan larutan NaCl 0.85% hingga
volume mencapai 4 mL
• Ambil 1 mL larutan sampel tersebut, masukkan ke dalam tabung reaksi
yang berisi 2 mL lautan 2xTSB, kemudian tutup rapat dan diberi label
• cara membuat larutan 2xTSB : 60 gr TSB + 20 gr NaCl dilarutkan dalam
aquadest 1 L, kemudian autoclave
• Inkubasi selama 18 jam pada suhu 30⁰C, kemudian bisa langsung proses
ekstraksi atau difiksasi menggunakan ethanol 96% dengan ratio (1:1) bila
tidak langsung diekstraksi (misal dikirim ke laboratorium atau tempat lain)
 PREPARASI SAMPEL

sampel segar ditimbang 1 gr + 500 ul 0,85% NaCl

dihancurkan

etanol (1:1) sampel dalam TSB (1:2), ink 18 jam homogenisasi dengan NaCl
hingga vol 4 ml

dipindah ke microtube sampel siap diekstraksi

 ISOLASI AHPND DARI ORGAN di TAMBAK
• Ambil sampel udang dengan jala sebanyak 5 ekor, gunting bagian karapaks
kepala dan ambil bagian stomach lalu masukkan ke dalam tabung reaksi
ukuran 10 mL yang berisi 3 mL larutan 1xTSB.
• Kemudian tutup rapat dan diberi label
• Inkubasi selama 6 jam pada suhu kamar, kemudian bisa langsung proses
ekstraksi atau difiksasi menggunakan ethanol 96% dengan ratio (1:1) bila
tidak langsung diekstraksi (misal dikirim ke laboratorium atau tempat lain)
PREPARASI SAMPEL AHPND
UNTUK MIKROBIOLOGY DAN PCR

air Organ : stomach

Plating media: TSA,

TCBS dan CAV
TERIMA KASIH
Non-Result/Technical Comparison

• Extraction procedure
• Amplification procedure
• Electrophoresis procedure
 COMPARATION FOR EXTRACTION AHPND
Test Quantity
Specimen
Nested CPF RT CPF AHPND AP4
<PL 15 10-15 PLs 10-15 PLs 20-25 PLs
Shrimp 30 mg 30 mg 20 mg
Mud 10 mg 10 mg 10 mg
Isolate in TSB 1 mL

• No significantly different from samples quantity

• On this evaluation we are focusing on shrimp and mud sample
 EXTRACTION PROCEDURE
Lysis Buffer Extraction Kit from CPF CTAB-DTAB IQ 2000

Step 1 : Lysis and DNA Binding 1. Prepare sample (PLs or stomach/hepato) on petri dish and dry remain ethanol
using towel tissue
1. Place specimen into a 1.5 mL microtube that contain 400 uL LB buffer 2. Place organ into 1,5 mL microtube containing 600 uL DTAB solution
2. Grind the sample in the tube with disposable grinder 3. Grind the sample in tube with disposable grinder and spin down
3. Centrifuge at 12000 rpm for 5 min 4. Incubate the sample at 75˚C for 5 menit, the cool down to room temperature
5. Add 700 uL Chloroform, vortex briefly then centrifuge at 12000 rpm for 5
4. Applay 200 uL of supernatant to the DNA column (yellow) minutes

6. Transfer 100 uL of the upper aqueous phase to new 1.5 mL microtube

5. Centrifuge at 12000 rpm for 30 sec to 1 min containing 100 uL CTAB solution and 900 uL aquabides , then vortex
6. Discard the flow through and absorb the remainder with a clean paper towels 7. Incubate the sample on waterbath at 75 ˚C for 5 menit
Step 2 : Washing 8. Cool down to room temperature, then sentifuge 12000 rpm for 10 menit
9. Carefully decant the supernatant, then 150 uL dissolve solution is added into
1. Add 500 uL of 75% ethanol into DNA column (1st) tube
2. Centrifuge at 12000 rpm for 30 sec to 1 min 10. Mix gently, spin down
3. Discard the-through and absorb the remainder with a clean paper towels 11. Incubate the sample on waterbath at 75˚C for 5 menit
4. Add 500 uL of 75% ethanol into DNA column (2nd) 12. Cool down to room temperature, sentrifuge 12000 rpm for 5 menit
5. Centrifuge at 12000 rpm for 30 sec to 1 min 13. Transfer 130 uL solution to new tube containing 300 uL of 96% ethanol
6. Discard the-through and absorb the remainder with a clean paper towels 14. Vortex briefly, sentrifuge 12000 rpm for 5 menit
7. Centrifuge at 12000 rpm for 3 min 15. Remove the supernatant, add 200 uL of 70% ethanol
8. Discard the collection tube and place the DNA column is each new clean 1.5 mL 16. Sentrifuge 12000 rpm for 5 menit, decant the supernatan
microtube
Step 3 : Elution 17. Dry pellet and dissolve in DEPC
1. Add 300 uL of pre-warm DI water to the center of each column
2. Let it stand for 2 min
3. Centrifuge at 10000 rpm for 3 min
4. Adding 100 uL DI water used fro DNA elution
 COMPARATION FOR EXTRACTION AHPND
Test Quantity
Item
Nested CPF RT CPF AP4
Avarage Concentration DNA 10-40 ng/uL 70-100 ng/uL
Purity 1.6-1.9 1.8-2.0
Method Lysis Buffer CTAB-DTAB
Estimation Time 35 Minutes 90 Minutes
DNA Column tube (to separate
Separate DNA using
DNA with another component
Tool reagent like chlorofom,
like protein,polisakarida)
dissolve solution

Result:
1. AP4 method give higher DNA concentration
2. Have same purity
3. AP4 method takes longer than the CPF method
4. CPF Extraction method use column tube

Note:
Both extraction method can be done for both PCR method
 PREMIX COMPOSITION

First PCR
Primer AHPND AP4 Primer AHPND Nested CPF Primer AHPND RT PCR CPF
reagent Volume/ reaksi (uL) reagent Volume/ reaksi (uL) reagent Volume/ reaksi (uL)
Nuclease water 9.5 Hotstart Mastermix 5.0 Fast Master Mix 7.5
Primer F779 0.5 First Primer mix 3.0 Primer mix 4.5
Primer R779 0.5 Probe Mix 1.0
Master Mix Go Green 12.5
Genom 2.0 Genom 2.0 Genom 2.0
Total Volume 25.0 Total Volume 10.0 Total Volume 15.0

Nested PCR
Primer AHPND AP4 Primer AHPND Nested CPF
Note Note
reagent Volume/ reaksi (uL) reagent Volume/ reaksi (uL)
Nuclease water 9.5 Hotstart Mastermix 7.5
Primer F176 0.5 Transfer 2.0 uL Nested Primer mix 7.5 Add 15 uL of nested
Primer R176 0.5 from first PCR reaction mixture
Master Mix Go Green 12.5 premix to to each tube after first
Genom 2.0 nested premix PCR was completed
Total Volume 25.0 Total Volume 15.0
 PCR CONDITION FOR AMPLIFICATION
First PCR RT PCR
Primer AHPND AP4 Primer AHPND Nested CPF
Temperature
Step Temperature ( °C ) Time Cycle Step Temperature ( °C ) Time Cycle Step Time Cycle
( °C )
1 94 2 min 1x 1 95 4 min 1x 50 2 min
Holding Stage 1x
94 30 sec 95 20 sec 95 20 sec
2 55 30 sec 30 x 2 58 20 sec 15 x 95 3 sec
Cycling step 50 x
72 90 sec 72 20 sec 60 30 sec
3 72 2 min 3 72 2 min
4 20 ∞ 4 20 ∞

Nested PCR
Primer AHPND AP4 Primer AHPND Nested CPF
Step Temperature ( °C ) Time Cycle Step Temperature ( °C ) Time Cycle
1 94 2 min 1x 1 95 4 min 1x
94 20 sec 95 20 sec
2 55 20 sec 25 x 2 58 20 sec 30 x
72 20 sec 72 20 sec
3 72 2 min 3 72 2 min
4 20 ∞ 4 20 ∞

Item RT CPF Nested CPF AHPND AP4

Amplification time 1h, 10m 1h, 25m 2h, 35m

 ELECTROPHORESIS RESULT
AHPND AP4 AHPND Nested CPF RT-PCR AHPND

In Lane 1 of the gel for the AP4 method,

there isa single amplicon band of 230 bp
from the 2nd (nested) PCR step indicating a Positive band of AHPND (toxin gene) are 232 bp
Result Analysis lowlevel of VPAHPND target in the template
and 354 bp
DNA. In Lane 2 of the gel for the AP4 method
there are 4 amplicon bands of 1269, 1142,
357 and 230 bp indicating a high level of
VPAHPND target in the template DNA.

Picture

Semi-Quantitative Semi-Quantitative
Result Quantitative
Give 3 different severity grade Give 3 different severity grade
characteristic (Copy number)
(light, medium, severe) (light, medium, severe)
 Result Comparison
• Validity result (sensitivity & specificity)
• Detection limit
• Consistency result
• Specificity to non-V.parahaemolyticus strain AHPND Plasmid
 1. Validity test
Table 2x2
Confirme Result by RT
Positive Negative Total
Positive 29 1 30
Test result by CPF Nested
Negative 24 20 44
Total 53 21 74

Sensitivity (%) 54.72

Specificity (%) 95.24

Confirme Result by RT
Positive Negative Total
Positive 30 0 30
Test result by AP4 Nested
Negative 23 21 44
Total 53 21 74

Sensitivity (%) 56.60

Specificity (%) 100.00

For validation test, RT PCR used as golden standard because the results are quantitative and has a
low detection limit.

1. Test sensitivity show that CPF method (54.72%) not significantly different with AP4 method (56.6%)
2. Test Specificity show that AP4 method (100%) slightly higher than CPF method (95.24%)
 2. Detection limit
First batch
DNA Concentration Real Time Result AP4 KIT CPF nested KIT
100 ng Positive 78239.2 Positive Positive
10 ng Positive 6184.9 Positive Positive
1 ng Positive 742.99 Positive Positive
100 pg Positive 70.01 Positive Positive
10 pg Positive 8.97 Negative Negative
1 pg Negative Undetect Negative Negative

Second batch
DNA Concentration Real Time Result AP4 KIT CPF nested KIT
100 ng Positive 152482.13 Positive Positive
10 ng Positive 13637.32 Positive Positive
1 ng Positive 1426.52 Positive Positive
100 pg Positive 177.99 Negative Positive
10 pg Positive 11.61 Negative Negative
1 pg Positive 3.39 Negative Negative

1. RT CPF has detection limit until 1 copy number

2. CPF nested has detection limit 100 copy number
3. AP4 method has detection limit 100 copy number
 3. Consistency result
No. of Sample Real Time Result AP4 KIT CPF nested KIT
1 Positive Positive Positive
2 Positive Positive Positive
3 Positive Positive Positive
4 Positive Positive Positive
5 Positive Positive Positive
6 Positive Positive Negative
7 Positive Positive Positive
8 Positive Positive Positive
9 Positive Positive Negative
10 Positive Positive Positive

Consistency result (% ) 100 100 80

Consistency test result show that CPF RT and AP4 method has 100%
consistency, but for CPF nested just have 80% consistency

Note:
Consistency will decrease if the sample has copy number in limit detection
 4. Specificity result to another non-EHP
plasmid
Item plasmid Real Time Result AP4 KIT CPF nested KIT
Positive control IQ 2000 WSSV Negative Undetect Negative Negative

Positive control IQ 2000 IMNV Negative Undetect Negative Negative

Positive control IQ 2000 IHHNV Negative Undetect Negative Negative

Positive control IQ 2000 TSV Negative Undetect Negative Negative

Positive control IQ 2000 YHV Negative Undetect Negative Negative

Positive control IQ 2000 GHV Negative Undetect Negative Negative

All three KIT can not react to the plasmid of WSSV, IMNV, TSV, YHV and
GHV
 Summary & Rekomendasi
• CPF Real time PCR adalah metode terbaik untuk
mendeteksi AHPND
• AP4 nested method tidak berbeda signifikan
dengan CPF nested dalam hal sensitivitas, tetapi
AP4 lebih baik daripada CPF dalam hal
specificity
• Kedua nested PCR method dapat digunakan
sebagai alat diagnosis untuk deteksi AHPND di
semua lab PCR CPP.