Shrimp Disease DNA Test Kit

1. Introduction

For shrimp farming, it is very important to watch the health conditions of shrimp continuously. Particularly, the shrimp farmer is threatened with major diseases, namely; AHPND / EMS Vibrio parahaemolyticus, IHHNV (Infectious Hypodermal and Haematopoietic Necrosis Virus), WSSV (White Spot Syndrome Virus) and EHP (Enterocytozoon hepatopenaei). Using the conventional method, these are checked individually which take a long time and need special techniques. “GenePasQ Shrimp Disease DNA Test Kits” was developed to detect these 4 diseases simultaneously. After PCR, you can visually judge the existence of the target genes with the PAS / DNA Chromatography method

2. Characteristic

◆ Detect 4 Shrimp diseases simultaneously.
・EHP (Enterocytozoon hepatopenaei)
・WSSV (White Spot Syndrome Virus)
・IHHNV (Infectious Hypodermal and Haematopoietic
Necrosis Virus )
・AHPND / EMS ( V. parahaemolyticus)
Electrophoresis is not needed.
Easy & Rapid
Judge the results by eyes (visually)

3. Shrimp Test Kit Contents (Good for 100 tests)

Be sure to close the PCR caps to avoid liquid leakage and evaporation during PCR. Then, perform PCR under the
following conditions. When you use a gene amplification machine and PCR enzyme other than our recommendation, set the conditions of PCR based on the following conditions. Modification of the amplification conditions may be needed depending on the PCR machine and enzyme.

5-3. Reaction using the DNA chromatography method

1) Take out “C) Latex solution” and “D) Dilution buffer” from refrigerator and keep at room temperature.
2) Prepare the Latex-working solution by diluting “C) Latex solution” 1:10 in “D) Dilution buffer”. Mix the solution uniformly with vortex mixer.
3) Centrifuge the PCR tubes after PCR briefly. Then, open the caps of PCR tubes after PCR and add 11 μL of the Latex
working solution. Mix the solution uniformly by pipetting.

4) Insert C-PAS (F8) in tube of step” 3)”. The opposite end of the absorbing pad must be inserted in the tubes.

※ Be sure to hold a part of absorbing pad of C-PAS(F8) with dried hands during use. Touching the parts other than the absorbing pad and holding it with wet hands may result in an insufficient reaction.
5) Leave them at room temperature for 10 minutes for developing. Perform the reaction at room temperature (20-30°C) and 40-80% humidity.
Caution: A low temperature and low humidity will cause False-positive detections.

5-5. Judgement of the results

Negative: When no target gene exists in sample, the blue line at the Tag7 (IC) line appears.
Positive: When target gene exists in sample, the blue line at the detection line appears.

Absorbing pad

Be sure to hold a part of the absorbing pad of C-PAS (F8) with dried hands during use.

・Position markers (Red lines)
The position of emerging lines is referenced against these Position Markers (Red lines). These Position Markers always appear with or without reaction.

・Flow control line
The blue line disappears when the solution passes through it.

・Detection line
A blue positive line appears at the detection line if the target genes exist in your sample.
The blue detection lines that exist before the reaction will temporarily disappear when the solution passes through. After that, the reaction line will appear again depending on the reaction. The blue lines that you see before using the C-PAS (F8) strip will gradually fade but these changes will have no effect on the reaction.

4. Equipment and reagents necessary for Shrimp Disease Test kits

4-1 Reagents

・Premix PCR Enzymes
We recommend the following reagents:
SYBR Premix ExTaq (Tli RNaseH Plus) (Takara)
Multiplex PCR plus Kit (Qiagen: 206152)
・RNase- Free Water

4-2 Instruments

・Gene amplification machine
We recommend the following units:
Quick Bath (ThermoGen)－ sold under GenePasQ trademark
GeneAmp9700 (ABI)
Veriti200 (ABI)

4-3 Other Instruments

・Vortex mixer
・Micropipettes

5. Protocol

5-1. Preparation of DNA solution

Please extract DNA from samples using your predetermined method. We recommend the following products
Qiagen DNA extraction kit, KOD FX Neo (TOYOBO).
GenCheck DNA extraction reagent (Fasmac)

5-2. Gene amplification by PCR

1) Prepare reaction mixture.
Add the following premix PCR solution to PCR tubes.
This Shrimp Disease Test kits are optimized for 10 μL of PCR system.

2) Add 1.0 μL of the sample DNA solution (Total 10 μL).
3) Amplify the nucleic acids by PCR.

* The IC (Internal Control) line often becomes faint when a
target gene is amplified.

WARRANTY
Nikken Biomedical Laboratory, Inc. makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made of standard quality. Buyer assumes all risk and liability resulting from the use of this product. There is no warranty of merchantability of the product or of the fitness of the product for any purpose. Nikken Biomedical Laboratory, Inc. agrees to replace any defective product but expressly disclaims liability for damages including special or consequential damage or expenses arising directly or indirectly from the use of this product.
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* This product uses a DNA chromatography strip: C-PAS (F8), which is produced by TBA, co., Ltd.; Patent licensed by NGK INSULATIORS, Ltd.

・Exclusive Distributor: SURE Marketing Company., Inc.
Suite 706 Metropolitan Terraces Condominium,
Sacred Heart Street, San Antonio Village,
Makati City 1203, PHILIPPINES
Phone: +63 2 897-24-30 up to 31
E-Mail: smci@sure-bio.com
URL: www.sure-bio.com
・By collaboration with TBA co., Ltd. (C-PAS supplier)
・Manufacturer: Nikken Biomedical Laboratory, Inc