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Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual


Catalog #: 1201
Reference #: 1201-01

BIOO Scientific Corp. Updated: September 24, 2009

TABLE OF CONTENTS
GENERAL INFORMATION .......................................................................................... 1

Product Description .................................................................................. 1 Summary of Kit Features .......................................................................... 2 Kit Contents, Storage and Shelf Life ......................................................... 3 Required materials for nucleic acid extraction, not provided with the kit 3 Required Materials for WSSV detection, not provided with the kit .......... 4 Safety Precautions .................................................................................... 4 Protocol for Extraction of Nucleic Acid from Shrimp .............................. 5
SHRIMP WSSV DETECTION KIT PROTOCOL ....................................................... 6

Guidelines for Assembling PCR Reactions............................................... 6 Avoiding Contamination in PCRs ............................................................. 6 Considerations for Choosing 1-Step vs 2-Step (nested) PCR Protocol .... 6 1-Step PCRs .............................................................................................. 7 PCR Thermal Cycling Protocol ................................................................ 8 2-Step Nested PCRs .................................................................................. 8 Analysis of Samples and Interpretation of Results ................................... 9
ADDITIONAL PROCEDURES .................................................................................... 10

Preparing and Running Agarose Gels .................................................... 10


TROUBLESHOOTING ................................................................................................. 12

Troubleshooting Nucleic Acid Extraction Step ....................................... 12 General Failure to See Expected Amplicons .......................................... 12 No Amplicon in Reactions Using WSSV Primers ................................... 13 Expected Amplicon Not Seen in Shrimp DNA Positive Control Reaction ................................................................................................................. 13 References ............................................................................................... 14
MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. MaxSignal is a registered trademark of Bioo Scientific Corporation (BIOO).

MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

GENERAL INFORMATION GENERAL INFORMATION Product Description


White Spot Syndrome Virus (WSSV) is claimed to be the most serious viral pathogen that affects cultivated shrimp worldwide (1). The MaxSignal Shrimp WSSV Detection Kit uses an end-point Polymerase Chain Reaction (PCR) assay to screen for WSSV in fresh or frozen shrimp or other potentially infectious material. The kit contains 2 pairs of oligonucleotide primers targeting nested regions of the WSSV VP466 gene (accession # EF564812), which encodes a structural protein located in the nucleocapsid (2). The WSSV amplicon produced using the WSSV-483 primers in the kit is 483 bp and the amplicon produced using the WSSV-388 primers is 388 bp. These WSSV amplicons have no significant homology to any other viral sequence. The detection limit for the synthetic WSSV template included in the kit is approximately 50 copies using the standard 1-step PCR protocol, and approximately 5 copies using the 2-step nested PCR protocol. For detection of WSSV in infected shrimp, positive signals have been obtained using the 1-step PCR protocol with input amounts of approximately 50 femtograms (50 x 10-15 gm) of total nucleic acid extracted from shrimp that were experimentally infected with high levels of the WSSV virus. The Shrimp Positive Control primers included in the kit were designed to the actin 1 gene (Accession # AF100986) from Penaeus monodon and will produce an amplicon of 249 bp from P monodon and also from Penaeus vannamei. The Shrimp Positive Control primers are used to demonstrate successful extraction of DNA that is able to support PCR. To maximize user convenience, the reagents for PCR (buffer, dNTPs, and thermostable polymerase) are provided as a 5x MasterMix. The MasterMix also contains components to allow the PCR to be directly loaded for gel analysis, without adding gel-loading solution. Input sample for this kit is total DNA or total nucleic acid extracted from solid tissue or derived from liquid tissues such as hemolymph; purification of viral particles for analysis is not required. The kit contains reagents for extraction of total nucleic acid from shrimp, for use as input sample in the PCR assay. The nucleic acid extraction protocol is designed for extraction of total nucleic acid (cellular and viral DNA and RNA) from small amounts (~10 20 mg) of solid tissue from shrimp or other samples to be tested, for example crayfish. The first step of the protocol is to grind or otherwise disrupt the tissue in Lysis Solution containing SDS and Proteinase K. The lysate is then incubated at high temperature to digest proteins and release nucleic acid, followed by removal of proteins and cellular debris by precipitation and centrifugation. The nucleic acid is then recovered from the clarified lysate by isopropanol precipitation. Yields of nucleic acid are typically between 10 100 g per prep. The nucleic acid recovered using this kit is especially useful for PCR. The RNA may not be of sufficient quality for applications that require highly intact RNA. The purity of the nucleic acid as determined by UV absorbance ratio (Abs 260 / Abs 280) is typically between1.8 2.0.

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

Summary of Kit Features


Kit contains reagents for 100 nucleic acid extractions from 10 mg 20 mg of tissue per prep. Kit contains reagents for 100 experimental PCRs (40 L per reaction) plus 10 additional PCRs that can be used for positive control reactions. PCR components are provided as a 5X Gel-Ready MasterMix. Forward + Reverse PCR primers are supplied as mixtures for user convenience. Sufficient amounts of each primer pair are provided for up to 100 PCRs, to allow maximum user flexibility in assay design. Precisely quantified WSSV positive control template is provided to verify kit performance and use in troubleshooting sample problems. PCR primers for amplification of shrimp DNA are provided to serve as endogenous positive control for successful DNA extraction. These primers can be used in multiplex reactions with the primers for WSSV detection. Molecular Weight marker is included for sizing of amplicons from experimental samples. Assay results are available in approximately 2.5 - 3 hours, depending on thermocycle profile.

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

Kit Contents, Storage and Shelf Life


Nucleic Acid Extraction reagents for up to 100 extractions from 10 20 mg of tissue are shown in the Table below.
Kit Component Lysis Solution Proteinase K Proteinase K resuspension buffer Protein Precipitation Solution Water for 75% ethanol Nucleic acid resuspension solution Volume per kit 50 mL 20 mg (2 x 10 mg) 1.5 mL 20 mL 15 mL (to be mixed with 45 mL ethanol) 5 mL Storage Conditions Room temp -20 deg (after resuspending) Room temp until use Room temp Room temp Room temp

The MaxSignal Shrimp WSSV Detection Kit contains reagents for 110 reactions of 40 L each as shown in the Table below. Store the kit at -20C. The shelf life is at least 12 months when the kit is properly stored.
Kit Contents 5X MasterMix - (complete with buffer, dNTPs, DuroTaq thermostable DNA polymerase, and gel-loading dye) WSSV-388 For + Rev PCR Primers - (5 uM concentration of each) WSSV-483 For + Rev PCR Primers - (5 uM concentration of each) Shrimp Positive Control For + Rev PCR Primers - (5 uM concentration each) WSSV Positive Control Template - (10,000 copies/L) Water - (Nuclease-free distilled water; packaged in 2 tubes) Molecular Weight Marker (MW Marker) - (BfuCI digest of pUC19 plasmid, 20 ng/L concentration) The 4 largest bands are 955 bp, 585 bp, 341 bp, and 258 bp. Standard amount to load is 20 L per lane. Amount 900 L 485 L 485 L 440 L 50 L 3.0 mL 200 L Storage -20C -20C -20C -20C -20C -20C -20C

Required materials for nucleic acid extraction, not provided with the kit
Ground glass conical homogenizer or other manual or mechanical tissue disruptor Heat block Microcentrifuge 1.5 mL microfuge tubes P-1000, P-200 range pipettors and tips Absolute ethanol (ACS grade or equivalent) Isopropanol (ACS grade or equivalent) Small container of ice

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

Required Materials for WSSV detection, not provided with the kit
Microcentrifuge Vortex mixer Thermalcycler Thin-wall PCR tubes (0.5 mL) and standard 1.5 mL or 2 mL microfuge tubes for assembling reactions Medium-volume pipettor and tips (eg P-200); note, barrier tips are recommended Low-volume pipettor and fine-bore tips (eg P-20); note, barrier tips are recommended Power supply and apparatus for running horizontal agarose gel Reagents for making and running agarose gel (see Additional Procedures) UV transilluminator Disposable gloves to prevent contact with kit components, tubes, and gel buffer Gel-documentation system or digital camera

Safety Precautions
Use standard precautions to avoid electrical shock when running gels, for example use gel boxes with secure lids and do not contact gel buffer during electrophoresis. Wear gloves and personal protective equipment to avoid contact with ethidium bromide, which is a mutagen. Do not look directly at gel under UV light, as this may result in damage to eyes. Wear safety goggles or view digital image of gel on a computer monitor.

BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product.
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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

Protocol for Extraction of Nucleic Acid from Shrimp


NOTE: this protocol is designed for use with 10 20 mg amounts of solid tissue. To process more tissue (up to ~ 50 mg), the reagent volumes may be scaled up 2-fold. In this case, transfer the lysate to a 2 mL tube at Step 3 and use two 1.5 mL tubes to precipitate the nucleic acid at Step 7. Before beginning the procedure Resuspend each tube of Proteinase K in 0.6 mL of Proteinase K Resuspension Solution. Vortex gently or flick tube to mix, then centrifuge briefly to collect liquid at bottom of tube. Store the Proteinase K solution at -20 deg C after use. Add 45 mL of 100% ethanol to the 15 mL of nuclease-free water provided to make a 75% Ethanol Solution. This may be stored at room temp. Prepare a heat block heated to 60 65 deg C.

Protocol 1. Excise 10 20 mg piece of tissue (about the size of uppercase letter O, 12-point-font), place in bottom of conical ground glass homogenizer, and add 0.5 mL Lysis Soln and 10 uL of Proteinase K. ( the volume of Lysis Solution should be at least 10 times the volume of tissue used). 2. Disrupt the tissue thoroughly by grinding for several minutes. The tissue lysate should be smooth and homogeneous. 3. Transfer prep to 1.5 mL microfuge tube and incubate for 30 min at 60 65 deg C. 4. Add 175 uL of Protein Precipitation Solution and vortex prep for ~ 20 sec. 5. Store prep on ice for 5 min. 6. Spin prep for 10 min, ~ 12,000 rpm in microfuge ( at 4 deg or room temp) 7. Remove ~90% of the clear supernatant to a fresh 1.5 mL microfuge tube and add 1.1 volumes isopropanol. Mix thoroughly by vortexing. The expected amount of supernatant to recover is ~ 0.7 mL. Add 0.77 mL isopropanol per 0.7 mL of supernatent. 8. Spin prep for 10 min, ~ 12,000 rpm, and then remove supernatant by carefully pouring off. A small white pellet is usually visible. 9. Add 0.6 mL of 75% EtOH, vortex vigorously to dislodge the pellet, and spin 1 min at ~ 12,000 rpm. 10. Double-aspirate supernatant (double aspirate means to decant or pipet off all visible fluid, blot rim to remove some residual fluid if decanted, then re-spin the tube for 5 - 10 sec to collect all residual fluid at bottom of tube, then use a fine-bore pipet tip to thoroughly remove the residual fluid).
11. Resuspend pellet in 150 uL Nucleic Acid Resuspension Solution, vortex ~ 5 10 sec, spin

briefly to collect liquid at bottom of tube, then heat ~ 5 min at ~ 55 - 60 deg C with intermittent vortexing to completely dissolve the nucleic acid. Store the prep at -20 deg C.

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

SHRIMP WSSV DETECTION KIT PROTOCOL SHRIMP WSSV DETECTION KIT PROTOCOL Guidelines for Assembling PCR Reactions
All kit components may be thawed at room temp or in a heat block not to exceed 37C. Avoid prolonged storage of kit components at elevated temperature during thawing. After thawing, mix all reagents (except water) by brief vortexing. Before opening tubes, pop-spin (centrifuge for a few seconds in a microcentrifuge) to collect all liquid at the bottom of the tube. Return the 5X Master Mix promptly to -20C freezer after use. The Tables shown in the Protocols section of this manual include examples of reagent volumes needed for running multiple PCRs. Please adjust the volumes of reagents used in your experiment to reflect the number of PCRs you plan to run.

Avoiding Contamination in PCRs


Do not assemble PCRs in the same room used for gel analysis of PCR reactions. When assembling reactions, use barrier pipet tips, and use tips that are long enough to avoid putting the shaft of the pipettor into the reaction tubes or kit component tubes. Use separate pipettors for assembling PCRs and for removing aliquots of post-PCR reactions for analysis. If possible store the pipettors, tips, racks, and tubes used for assembling PCR reactions under a UV hood when not in use. Store the post-PCR component (MW marker) in a separate location from the other kit components. Always include no-template negative control(s) to rule out non-specific amplification (i.e. contamination). When using the water in the kit to dilute the WSSV Positive Control Template, remove an aliquot of water to a separate tube to minimize chance of contamination. If desired, the positive control template can be diluted using water or other diluent from another source.

Considerations for Choosing 1-Step vs 2-Step (nested) PCR Protocol


The 1-step protocol requires less time to carry out and is sufficient to detect WSSV virus in most cases. The 1-step protocol has been used to detect WSSV in infected shrimp when the nucleic acid was diluted more than one-million-fold, and is able to detect synthetic WSSV target down to a level of approximately 50 copies. The 2-step protocol is typically ~10 fold more sensitive than the 1-step protocol, however it has the disadvantages of being more prone to contamination and requiring a longer time to complete the assay. Risk of contamination may be reduced by using barrier tips, keeping the pipettors under UV light, and using UV-sterilized work surface. Risk of contamination is also reduced when the frequency of positive samples in an experimental run is low. Many studies have been carried out to determine WSSV detection rates using 1-step vs 2-step PCRs for samples of different developmental stages, different stages and levels of infection, and taken from different environmental and anatomical sites (see for example ref 3-5). Detection via 1-step vs 2-step PCR has been used as a rough estimate of the level of WSSV infection, where samples that are positive in 1-step PCR are considered to have high levels of WSSV and samples that are positive only after 2-step PCR have low-level infection.
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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

A reasonable strategy may be to use the 1-step protocol for routine screening of individual shrimp or small larvae collected under conditions where WSSV levels are expected to be easily detectable. Use the 2-step protocol for challenging situations, for example when very low-level infection is expected or when large pools of samples or highly dilute samples are being assessed.

1-Step PCRs
1. Assemble the components as shown in Table 1. After all components have been added, vortex the tube vigorously (~ 15 sec) to thoroughly mix the components, then pop-spin and store at room temp until use. Do not place the mixture on ice. This may cause it to partially solidify, which will lead to suboptimal results, even if it is thawed before dispensing into the PCR tubes.
TABLE 1 Component Sample (DNA, total nucleic acid, or Positive Control Template) 5X Master Mix WSSV-483 PCR primers Shrimp Positive Control PCR primers (optional) Nuclease-free water Amount per 40 L PCR Typically 3-5 L (~30 300 ng of nucleic acid) 8 L 4 L 4 L to give final reaction volume of 40 L Amount for 10 Reactions (not added to mix) 80 L 40 L 40 L (volume per reaction) x 10

2. Add appropriate volume of above mixture to each tube to give final volume of 40 L (this will be 35 L if 5 L of sample nucleic acid is used per reaction). 3. Add sample (DNA, total nucleic acid, or cell-free fluid such as hemolymph, or WSSV Positive Control Template) to each tube. Typically ~30 300 ng of nucleic acid is used per reaction. For the WSSV positive control add 5 L of WSSV Positive Control Template, which may be diluted from the initial concentration of 10,000 copies/L if desired, using the water provided with the kit or other nuclease-free diluent. Take precautions to avoid contaminating the kit components with the WSSV Positive Control Template. Always include a no-template negative control reaction in the assay.
4. Vortex samples briefly and pop-spin each tube to collect contents at bottom of tube, then

place into thermalcycler and run preset program as suggested below.

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

PCR Thermal Cycling Protocol


Program your thermalcycler according to the suggestion below or use an alternative program based on previous experience. If your instrument does not have a heated lid, overlay the reactions with mineral oil before running the PCR to prevent evaporation. Note: Since thermalcyclers differ in parameters such as ramp times needed to reach target temperatures, some empirical optimization of thermal cycling profiles may be needed to achieve maximum sensitivity for WSSV detection using this kit. Recommended profile for 1-step PCR:
1. 2. 3. 4.

Hold 3 min at 94C (initial denaturation step) Cycle 35x : 94C, 30 sec / 55C, 30 sec / 72C, 30 sec (cycling step) Hold 5 min at 72C (final extension step) Hold indefinitely at 4C or 18C (hold reactions at low temp until analysis) Store reactions at -20C prior to running.

2-Step Nested PCRs


This protocol may be used for detection of very low levels of WSSV viral infection. A first-round preamplification PCR using the WSSV-483 primers is carried out for 20 - 25 cycles, and an aliquot of the first-round PCR product is then used as template for a second-round PCR, using the WSSV-388 primers. The WSSV-388 primers have binding sites that are within the 483 bp amplicon produced in the first-round PCR. First-round PCR: Assemble as shown in Table 1. Place tubes into thermalcycler and run the preset profile for 20 - 25 cycles as described in previous section. Second-round PCR:
1. Assemble the reaction mixture for the second-round, nested PCR as shown in Table 2 and

add 35 L to each prelabeled second-round PCR tube.


TABLE 2 Second-round PCR Components 5X Master Mix WSSV-388 For + Rev PCR primers Nuclease-free water Amount per 40 uL PCR 8 L 4 L 24 L Amount for 10 reactions 88 L 44 L 264 L

2. After the first-round PCR is finished, remove tubes and pop-spin to collect all liquid at bottom of tube. Using barrier tips, remove 2 - 5 L of each first-round PCR and add to the corresponding pre-labeled second-round PCR tube containing 35 L of second-round reaction mixture. Use a separate barrier pipet tip for each sample. For the second-round PCR negative control, add 5 L of the no-template negative control from the first-round PCR.
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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

3. Place tubes into thermalcycler and run preset profile as described above. For 2-step nested PCR, 20 - 25 cycles are typically used for the first-round PCR and 25 for the second-round PCR.

Analysis of Samples and Interpretation of Results


To minimize chance of contamination, it is recommended that the post-PCR tubes not be opened in the same area as used to set up and run the reactions. Run ~ 20 - 25 L of each PCR on a 2% native agarose gel (protocol for making and running the agarose gel is outlined in the Additional Procedures section). It is not necessary to add gel-loading solution to the samples, since the 5X Master Mix already contains tracking dyes and components to cause the sample to sink to the bottom of the wells. The tracking dyes will separate into blue and yellow bands during electrophoresis. Load 20 L (400 ng) of the MW marker to aid in determining the size of amplicons in the experimental samples. The MW marker contains tracking dye and is ready to load directly. The sizes of the 4 largest bands in the MW marker are 955 bp, 585 bp, 341 bp, and 258 bp. The smaller bands, which may not be visible, are 141 bp, 105 bp, 78/75 bp, 46 bp, and 36 bp. Interpretation of results from experimental samples: Samples that are positive for WSSV infection will show a band of 483 bp from 1-step PCR. Samples that are positive using the 2-step PCR will show a band of 388 bp. The 2-step PCRs may show both the 483 bp and 388 bp bands, especially when the input sample had high levels of WSSV (~10,000 copies or greater). Interpretation of Results from WSSV Positive Control Reaction Reactions containing 50 50,000 copies of WSSV Positive Control Template should show bands of 483 bp or 388 bp, depending on whether WSSV-483 or WSSV-388 primers were used. Note, 50 copies corresponds to 5 L of 1:1,000 dilution and 50,000 copies corresponds to 5 L of undiluted Positive Control Template. The detection limit depends partly on the efficiency of the thermalcycler used. Interpretation of Results from Shrimp Positive Control Reaction Nucleic acid samples extracted from tissues that are either positive or negative for WSSV infection will typically show a band of 249 bp when used as template with the Shrimp Positive Control primers. Note: The Shrimp Positive Control primers are not expected to yield a product if the template consisted of a cell-free extract such as hemolymph or an environmental sample such as pond water. The Shrimp Positive Control primers will not produce a product from the WSSV Positive Control Template. Note: The WSSV-388 or WSSV-483 amplicon may be produced even in the absence of the 249 bp shrimp positive control amplicon. This can happen if the sample is highly infectious, resulting in many more copies of WSSV target than shrimp genome target in the input sample, or if the sample genomic DNA is highly degraded. Be sure to compare amplicon sizes to MW marker bands to distinguish WSSV amplicons from shrimp genomic amplicons. This is especially important if the WSSV primers were
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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

multiplexed with the Shrimp Positive Control primers. Non-Specific Amplification Non-specific amplification (primer dimer) is typically seen as smaller, more diffuse ethidium-staining material that usually migrates behind the yellow tracking dye band. Sometimes non-specific ethidium-stained material is also observed in the wells; this does not constitute a positive result for viral infection. Primer dimer products may be more pronounced for the WSSV-388 primers than for the WSSV-483 primers.

ADDITIONAL PROCEDURES ADDITIONAL PROCEDURES Preparing and Running Agarose Gels


For analysis of the relatively short amplicons produced by the primers in this kit, 2% agarose gels are recommended for optimal resolution. The recommended gel running buffer is Tris-Borate-EDTA (TBE), although Tris-Acetate-EDTA (TAE) may also be used. Reagents needed to make and run a 2% agarose gel in TBE buffer: 1. 10X TBE buffer (composition of 1X TBE is 89 mM Tris-borate pH 8.3 / 2 mM EDTA). Chemicals may be purchased from Sigma Life Sciences: Tris-base cat #T1503; boric acid cat #B6768; EDTA disodium salt catalog # ED2SS. To make 1 liter of 10X TBE dissolve 108 gm Tris base, 55 gm boric acid, and 40 mL of a 0.5 M solution of EDTA, in distilled water to a final volume of 1 liter. The pH should be 8.3. Store at room temp. To make 100 ml of 0.5 M EDTA, dissolve 18.6 gm disodium EDTA in ~ 70 mL distilled water, pH to 8.0 with NaOH (EDTA will not dissolve until pH is approximately 8), then adjust to final volume of 100 mL with distilled water. 2. Agarose powder, for example purchased from Sigma Life Sciences: cat # A-9539 3. Distilled deionized water (does not need to be sterile or autoclaved).
4. Ethidium bromide (EtBr), 10 mg/mL solution in distilled water. Dry powder may be

purchased from Sigma Life Sciences: ethidium bromide, cat # E8751. Ethidium bromide is also available as a liquid stock solution from several companies, for example FLUKA cat #46067. Protocol for Making 2% Agarose Gel: Make 1 liter of 1x TBE with ethidium bromide (1x TBE/EtBr) by mixing 100 mL of 10x TBE (see above) with 900 mL distilled water and 50 L of 10 mg/mL ethidium bromide stock solution. Use the 1x TBE/EtBr to make the gel and to run the gel. To make 100 mL of 2% agarose gel, add 2 gm agarose to 100 mL 1x TBE/EtBr in a 250 mL beaker or similar container, swirl to mix, cover loosely with plastic wrap, then microwave for ~ 1 2 min on High power to completely melt the agarose. To aid in melting the agarose, stir the mixture gently once or twice during the microwave heating. The solution should be clear and homogeneous when the agarose has completely melted. After melting, cool the agarose to ~
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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

60C or until the beaker is cool enough to hold in your hand (it should still be quite warm). Prepare the gel tray if necessary by taping the ends. To avoid leaks, melted agarose can be applied along the seam between the tape and the edge of the tray (especially at the corners) and allowed to solidify before pouring the gel. Pour the melted agarose into the prepared gel tray which already contains the comb and allow it to solidify. It will become slightly cloudy when solidified. Do not remove the comb until the agarose has completely solidified. Adding some buffer to the top of the solidified gel around the comb before removing it may help to avoid tearing the wells when the comb is removed. Be sure to remove any tape used to seal the ends of the gel tray. Once the comb has been removed, place the gel into the electrophoresis box and add buffer (1xTBE/EtBr) to a level high enough to fill both reservoirs and completely submerge the gel to a depth of a few millimeters. Check the wells to make sure there are no pieces of loose agarose; if so, they can usually be removed by gently flushing the wells with buffer. If the gel will not be run immediately, the apparatus containing the gel and buffer can be covered with plastic wrap and stored at room temp. Protocol for Running Agarose Gel: 1. After the gel and buffer are placed in the electrophoresis box, connect the leads (the red and black insulated wires) to the power source and to the gel box. The black lead is usually connected to the top or left-side electrode of the gel box and the red lead connected to the bottom or right-side electrode. The other ends of the leads are connected to the corresponding color-coded ports on the power supply. It is recommended to check the gel and power source before loading the samples to verify that it is running properly. Set the power source to run constant voltage. Place the lid on the electrophoresis chamber and turn on the power. To make sure that current is flowing through the gel, observe the apparatus from the side and look for small bubbles to form and rise from the wire that is in the chamber connected to the black lead (typically this is the top or left-hand chamber). Be sure to turn off the power before loading the gel. 2. The gel should be placed into the electrophoresis chamber and submerged with 1x TBE-EtBr running buffer before loading. Use a medium-volume pipettor (eg P-20 or P-200 or equivalent) to carefully load samples into the wells. Typical volume of sample to load is 20 - 25 L. Load 20 L of Molecular Weight marker (MW marker) provided with the kit (note, the MW marker has already been mixed with Gel Loading Solution). To minimize chance of PCR contamination, use a dedicated pipettor to load the PCR products (do not use the same pipettor used to set up PCRs). Run the gel at constant voltage, typically about 80 100 volts, until the blue tracking dye band has migrated about 2 3 cm from the wells (this usually takes about 30 min).
3. Turn the voltage down to the lowest setting, then turn the power supply off. Remove the

gel and place it on an ultraviolet (UV) transilluminator. Examine the gel under either long-wave or short-wave UV light for presence of products of the expected size (see Analysis of Results section). To avoid eye injury, be sure to wear UV-protective safety glasses or a UV-protective face mask when examining the gel directly. As an alternative to visualizing the gel directly, it may be digitally photographed and examined on a computer monitor to avoid operator exposure to UV light.

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

Suggestions for Apparatus for Running Agarose Gels for Detection of Short Amplicons For separation and detection of the relatively short amplicons produced by this kit, we recommend using gels and gel boxes that are short and wide. The advantage of using short gel boxes is that by minimizing the distance between the anode and cathode to approximately 16 cm (~ 6 inches), lower voltage is required for the amplicons to migrate a given distance. This maximizes the number of hours that the running buffer can be used and saves wear on the power supply. The advantages of using wide gels are that more samples can be run in adjacent lanes, which makes it easier to compare the results of multiple different samples and minimizes the number of molecular weight marker lanes that are needed. Also, the gels can typically be re-used several times after running the amplicons off the end of the gel, and short gels facilitate this process. The suggested dimensions for the gel are 5 - 6 cm long by 20 22 cm wide; this will allow 30 samples to be analyzed on a single gel if using a comb that forms wells which are ~ 5 mm wide with ~1 mm partition between each well. For a schematic diagram of the gel box, gel tray, and comb recommended for routine analysis of short amplicons such as those produced using the primers in this kit, please contact BIOO Scientific technical support at techsupport4@biooscientific.com.

TROUBLESHOOTINGTROUBLESHOOTING Troubleshooting Nucleic Acid Extraction Step


The most common problem is inhibitors of PCR recovered with the nucleic acid. This can usually be overcome by either using 10-fold less nucleic acid in the PCR, or by using less tissue as starting material in the prep. Sometimes problems are caused by pigments or inhibitors in specific tissues, for example eyeball preps may have a dark pigment that co-purifies with the nucleic acid. Another problem is degraded nucleic acid. Degradation is usually due to a long lag time between harvesting the tissue and starting the prep, or due to repeated freeze-thaw of frozen tissue. To avoid degradation, disrupt the tissue as soon as possible after harvesting or store it in a reagent designed to preserve nucleic acid in solid tissue. Alternatively, harvested tissue may be snap-frozen and stored at -70 deg C. Storage at -20 deg C may result in gradual decline in nucleic acid integrity. Note however that degraded nucleic acid is usually adequate for use as template in PCR, especially for amplicons shorter than ~ 500 bp. For additional questions please contact BIOO Technical Services at: Techsupport4@biooscientific.com.

General Failure to See Expected Amplicons


Possible Causes
Ethidium bromide was not added to the gel and running buffer. Gel was visualized on tray that was not UV transparent. Thermalcycler was not programmed correctly or PCR run was interrupted, or your instrument

Recommended Action
Verify that the MW marker can be visualized when the gel is placed directly on the UV transilluminator. If not, re-make the buffer and add EtBr according to protocol and re-run the samples and/or MW marker. Determine whether the MW marker can be visualized if the gel is placed directly on the UV transilluminator. If so, problem may have been due to placing gel on non-UV transparent tray for analysis. Change apparatus to use UV-transparent tray OR place gel directly on UV transilluminator for analysis. Determine PCR run history if appropriate software is included with your instrument. Run the WSSV positive control reaction and if it does not show expected amplicon, run a PCR using the kit components with your own positive
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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01
requires different settings from those suggested in this manual. control template DNA and PCR primers to produce an amplicon of ~350 550 bp (similar in size to the amplicons from the kit controls). Use the thermalcycler profile for WSSV as described in the manual, to determine whether these settings are appropriate for making 350 500 bp amplicons using your thermalcycler. Verify that 10x Gel Running Buffer was diluted to 1X for making and running gel and that agarose was completely melted. Run MW marker and verify ability to detect at least the 4 largest bands (955 bp, 585 bp, 341 bp, and 258 bp). Follow the protocol carefully and repeat the assay. Check records to see how many times the kit has cycled from the refrigerator. Check to see if the kit was left at extreme temperatures for too long. Verify the kit was not used after its expiration date.

Gel was made or run incorrectly. Operator error in assembling or running the PCRs. Excessive kit stress has occurred or kit was used beyond expiration date.

No Amplicon in Reactions Using WSSV Primers


Possible Causes
Sample is negative for WSSV infection. WSSV primers were degraded.

Recommended Action
Use the WSSV Positive Control Template and WSSV-483 and/or WSSV-388 Primers in a WSSV positive control reaction to verify performance of the kit. Use WSSV Positive Control Template with the other primer WSSV pair to verify kit performance.

Expected Amplicon Not Seen in Shrimp DNA Positive Control Reaction


Note: The WSSV-388 or WSSV-483 amplicon may be produced even in the absence of the 249 bp shrimp positive control amplicon. This can happen if the sample is highly infectious, resulting in many more copies of WSSV target than shrimp genome target in the input sample. Be sure to compare amplicon sizes to molecular weight marker bands to distinguish WSSV amplicons from shrimp genomic amplicons.

Possible Causes
Problem with storage of shrimp samples prior to nucleic acid extraction.

Recommended Action
Analyze extracted nucleic acid on agarose gel to determine intactness and yield. To minimize degradation and improve yield, use tissue from freshly sacrificed shrimp (i.e. excise the tissue sample as soon as possible after the shrimp is removed from its habitat). If using frozen samples, ensure that shrimp was snap-frozen immediately after being removed from its habitat, and that it was maintained at a temperature of -20 C or colder until use. Analyze extracted nucleic acid on agarose gel to determine intactness and yield. To minimize degradation and improve yield, do not allow frozen samples to thaw for long period of time or at elevated temperature. Use extraction method known to yield high-quality DNA. Dilute template and repeat the PCR. Optimal range of nucleic acid to add per 40 uL PCR is approximately 30 ng 300 ng. Mix some of your shrimp sample nucleic acid with 10,000 copies (1 uL) of the WSSV positive control template provided with the kit and run the positive control PCR using WSSV-388 or WSSV-483 primers. Include a reaction containing only the WSSV positive control template as a control for the PCR. If the WSSV amplicon is observed in the absence but not the presence of your shrimp nucleic acid sample, this indicates the presence of PCR inhibitors in your sample. Dilute your sample 1:10 or 1:100 and re-run the assay. Optimal input range of nucleic acid is approximately 30 ng 300 ng. No shrimp genomic DNA is present in your sample to produce shrimp positive control amplicon, so the result is as expected. Shrimp positive control primers were designed to detect Penaeus monodon or Penaeus vannamei or Littopenaeus vannamei; if none of these produce the expected amplicon your shrimp sample may be from a different species. Contact BIOO Scientific technical support for assistance in designing positive control primers to detect genomic DNA amplicon in your shrimp species.
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Problem with extraction of nucleic acid from samples. Too much nucleic acid was added to the PCR. Contaminants causing PCR inhibition were recovered during extraction of nucleic acid from your shrimp sample. Not enough template DNA was added to the PCR. Sample was non-cellular fluid such as hemolymph or pond water. Sequence difference between your shrimp species and species used to design shrimp positive control primers.

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MaxSignal Shrimp Nucleic Acid Extraction and WSSV Detection Kit Manual 1201-01

References
1. Escobedo-Bonilla CM, Alday-Sanz V, Wille M, Pensaert MB, and Nauwynck, HJ A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus. Journal of Fish Diseases 31:1-18 (2008). 2. Tsai JM, Wang HC, Leu JH, Wang AHJ, Zhuang Y, Walker PJ, Kou GH, and Lo CF Identification of the nucleocapsid, tegument and envelope proteins of the shrimp white spot syndrome virus virion. Journal of Virology 80:3021-3029 (2006). 3. Thakur PC, Corsin F, Turnbull JF, Shankar KM, Hao NV, Padiyar PA, Madhusudhan M, Morgan KL, Mohan CV. Estimation of prevalence of white spot syndrome virus (WSSV) by polymerase chain reaction in Penaeus monodon postlarvae at time of stocking in shrimp farms of Karnataka, India: a population-based study. Diseases of Aquatic Organisms. 49(3):235-43 (2002). 4. Withyachumnarnkul B. Results from black tiger shrimp Penaeus monodon culture ponds stocked with postlarvae PCR-positive or -negative for white-spot syndrome virus (WSSV). Diseases of Aquatic Organisms 39:21-27 (1999). 5. Hsu HC, Lo CF, Lin SC, Liu KF, Peng SE, Chang YS, Chen LL, Liu WJ, Kou GH. Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders. Diseases of Aquatic Organisms 39(1):13-9 (1999).

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