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