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Laboratory Procedure OPFLP-09
HEALTH PRODUCTS AND FOOD BRANCH
Concentration and detection of hepatitis A virus from contaminated strawberries by the pathatrix system and reverse transcription polymerase chain reaction
Kirsten Mattison, Michelle Plante, Sabah Bidawid
Health Canada, Bureau of Microbial Hazards, 251 promenade Sir Frederick Banting Driveway, Ottawa, ON, Canada,K1A 0K9
Microbiological Methods Committee
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204E
HPFB, Ottawa, Ontario, K1A 0K9
This method is applicable to the concentration and detection of hepatitis A virus (HAV) from food samples. This method will not differentiate between infectious and non-infectious viruses.
In test sensitivity studies, this method was used to consistently detect 1000 PFU HAV in artificially-inoculated strawberries.
The Pathatrix™ machine forcefully circulates non specific positively charged magnetic beads throughout berry samples. The cationic beads interact with the negatively charged viral capsids and the two are captured onto a magnet. Following subsequent washes with PBS buffer, the beads are resuspended in PBS and viral RNA is extracted with the QIAamp™ viral RNA mini kit. Extracted RNA is amplified by RT-PCR and amplicons are visualized by gel electrophoresis.
4.1 See Appendix A of Volume 3.
5.1 The number of samples will have to be determined on the basis of the client needs (e.g. data collection or surveys) or investigational purposes (e.g. Outbreaks).
The Laboratory Supervisor must ensure that completion of the analysis, described in this method, must be done in accordance with the International Standard referred to as "ISO/IEC 17025:1999 (or latest version). General requirements for the competence of testing and calibration laboratories".
6.1 Thermal cycler (Eppendorf Mastercyler Gradient or equivalent).
6.2 Microfuge 22R refrigerated tabletop centrifuge (Beckman Coulter or equivalent).
6.3 Microwave oven or hot plate.
6.4 Submarine gel casting tray and buffer reservoir, power pack and an appropriate comb.
6.5 Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels.
6.6 Photo documentation system (optional, for photographic records), including polaroid camera (hand-held or fixed), hood and Polaroid 667 film or equivalent.
6.7 Adjustable micropipettors: to cover range of volumes: 0.5 to 10 μl, 10 to 100 μl, and 100 to 1000 μl with specific filtered pipet tips
6.8 Waterbath capable of maintaining a temperature of 40-50°C.
It is the responsibility of each laboratory to ensure that the block heaters or water baths are maintained at the recommended temperatures. Where 37°C is recommended the waterbath may be at 37°C +/- 1°C. For all other temperatures it may be +/- 2°C.
6.9 Freezers capable of maintaining - 20°C and - 80°C.
6.10 Pathatrix™ or Pathatrix-ULTRA™ workstation (Matrix Microscience Inc.).
6.11 Magnetic wash tube rack (Matrix Microscience Inc.).
6.13 Container for Ice.
6.14 Vortex mixer.
6.15 Pre-sterilized plastic and rubber disposable kits (Matrix Microscience Inc.).
6.16 Microfuge tubes - 2.0 and 1.5 ml capacity.
6.17 Tubes for PCR - thin wall 0.2 ml capacity (depending on thermal cycler model).
6.18 Custom magnetic silica particles cationally/positively charged (Matrix Microscience Inc. product number PCAT50, no substitutions).
6.19 Medium disposable weigh boats.
6.20 Stomacher bags with filter.
6.21 Feline Calicivirus strain F9 (American Type Culture Collection product number VR-782, no substitute).
6.22 QIAamp™ viral RNA extraction kit (product number 52904 or 52906, no substitutions).
6.23 Qiagen® OneStep RT-PCR Kit (product numbers 210210 or 210212, no substitutions).
6.24 Agarose (molecular biology grade).
6.25 DNA Ladder 100 bp (or equivalent).
6.26 Ethidium bromide (molecular biology grade) or SYBR Safe dye (Invitrogen product number S33102 or equivalent).
6.27 Rnase inhibitor.
6.28 Boric acid (molecular biology grade).
6.29 Tris[hydroxymethyl]aminomethane (e.g., Tris base molecular biology grade).
6.30 Ethylene Diamine Tetra Acetic acid (EDTA) disodium salt (molecular biology grade).
6.31 Glycine, non-animal source (molecular biology grade).
6.32 Sodium Chloride (molecular biology grade).
6.33 NaOH (ACS grade or better).
6.34 HCl (ACS grade or better).
6.35 Water DNAse RNAse free (molecular biology grade).
6.36 Phosphate buffered saline pH 7.2 (Invitrogen product number 20012-027 or equivalent).
6.37 Glycine buffer (See section 10.1 for preparation).
6.38 5X Tris-Borate-EDTA (TBE) buffer (See section 10.3 for preparation).
6.39 Ethanol 95- 99%.
6.40 Houde/Guévremont primers (SH-Prot-A and SH-Prot-1) for the detection of hepatitis A virus (See section 9.1.1 for DNA sequences).
6.41 FCV primers (CBK-1 and CBK-2) for the detection of feline calicivirus (FCV) (See section 9.1.2 for DNA sequence).
7.1.1 During storage and transport keep the sample units refrigerated (1 to 5°C).
7.1.2 Analyze the sample units as soon as possible after receipt at the laboratory.
7.2.1 Prepare all buffers and solutions as described in section 10.
7.2.2 Prepare and verify the suitability of all controls. Each test sample will be spiked with 105 plaque forming units (PFU) of the feline calicivirus (FCV) internal processing control. One negative processing control (uninoculated strawberries purchased locally) will be run in parallel with each set of up to four test samples. Hepatitis A virus (HAV) and FCV RNA must be available for use as PCR positive controls.
7.2.3 Measure twenty-five grams of strawberries onto a disposable weigh boat (up to a maximum of four test samples and one negative process control).
7.2.4 Add 105 PFU of FCV to each test sample (up to a maximum of four test samples). It is recommended that the viral stock be diluted in PBS such that the inoculation can be performed in a 100 μl volume.
Note: A compact disc containing a video demonstration of the use of the Pathatrix™-ULTRA is available from the manufacturer. This video is also avalable on the Matrix Microscience Inc. website. It is recommended that this video be viewed prior to sample processing.
7.3.1 Switch on the Pathatrix™ workstation.
7.3.2 Transfer each twenty-five gram sample of strawberries into a separate stomacher bag.
7.3.3 Add 225 ml of glycine buffer to each stomacher bag and shake samples by hand for approximately 30 sec.
7.3.4 Add 1.55 mL of 1N HCl to the sample + buffer in the stomacher bag and shake samples by hand for approximately 30 sec.
7.3.5 Place each stomacher bag into a separate incubation pot on the Pathatrix machine.
7.3.6 Aseptically remove the sterile disposable kit from the bag and ensure that the pot lid is oriented such that tubing extends from the centre and left of the lid (The nine o’clock position).
7.3.7 Hold the wash vessel in your left hand and place this into the holder. Release the phase and, using both hands insert the pot lid into the stomacher bag. Secure the phase within the phase holder and locate the elution pipette under the retainer.
7.3.8 Load the inlet tubing into the pump head and take care when closing the pump head to avoid trapping the tubing.
7.3.9 Vortex the vial of capture beads for a minimum of 10 seconds immediately prior to dispensing. Ensure that the beads are fully resuspended.
7.3.10 Disconnect the large tubing from the front of the valve that is also connected to the wash vessel and aseptically add 50 μl of capture beads into the tubing. Reconnect the large tubing to the front valve.
7.3.11 When loading more than one sample it is advisable to vortex the capture beads each time prior to dispensing.
7.3.12 Fill each wash vessel with 100 ml of PBS.
7.3.13 Set the temperature of each unit to 20-25°C and set the timer to 30 min.
7.3.14 Press the “RUN” button, recirculation will start immediately. Firmly push the pot lid down. Note that the timer will only begin when the test temperature is reached.
7.3.15 When completed the system displays the wash ready symbol.
7.3.16 Twist the tab on the side of the plunger on the front valve until it breaks off. Using your first finger and thumb press the plunger down until it reaches a natural stop.
Note: Excessive force is not required to depress the plungers on the consumables.
7.3.17 Press “RUN” on the key pad to initiate wash step. Press “RUN” a second time to stop the pump before air enters phase.
7.3.18 Twist the tab on the side of the plunger on the rear valve until it breaks off. Using your thumb and first finger press the plunger until it reaches a natural stop. Note excessive force is not required.
7.3.19 Open the pumphead and carefully remove the phase from the magnet.
7.3.20 Using the pipette attached to the rear valve gently flush the wash buffer over the phase, a minimum of 5 times.
7.3.21 Remove the wash vessel lid with internal tube still attached from the wash vessel and allow the liquid to drain back into the wash vessel.
7.3.22 Discard stomacher bag and disposable kit as biohazardous waste and place the flat lid provided on the wash vessel.
7.3.23 Place wash vessel into the wash vessel rack and leave to stand at room temperature for 5 minutes for the beads to be drawn to the magnet.
7.3.24 Aseptically remove all of the liquid from the wash vessel taking care not to remove any of the beads.
7.3.25 Resuspend the beads by a brief vortex in 140 μl of PBS and transfer each sample to a sterile 1.5 ml microfuge tube.
Note: All reagents used in this section are provided in the kit with the exception of cold ethanol. The protocol for nucleic acid extraction from viruses has been established according to the manufacturer’s instructions provided in the kit manual. It is important that once the carrier RNA has been added to the AVL buffer, it be stored at 4°C. Also, AVL buffer must be completely thawed to dissolve any precipitate prior to being used.
7.4.1 Samples consisting of beads in 140 μl of PBS are ready in 1.5 ml microfuge tubes from 7.3.24. If positive controls for HAV and FCV RNA are needed, 140 μl of viral stock may be placed in a 1.5 ml microfuge tube and processed in parallel with the test samples.
7.4.2 Add 560 µl of buffer AVL to each sample.
7.4.3 Vortex tubes for 2 seconds.
7.4.4 Incubate the samples for 10 min at room temperature (15 - 25°C).
7.4.5 Add 560 µl of ethanol to the samples.
7.4.6 Vortex tubes.
7.4.7 Transfer 630 µl of the solution to a QIAamp spin column provided in the kit.
7.4.8 Close the caps and centrifuge the samples at 6,000 x g (8000 rpm) for 1 min.
Note: Each centrifugation step is carried out at room temperature.
7.4.9 Transfer each spin column to a clean 2 ml collection tube provided in the kit.
7.4.10 Discard the previously used collection tubes.
7.4.11 Transfer the remaining 630 µl of each solution to its respective QIAamp spin column.
7.4.12 Centrifuge at 6,000 x g (8,000 rpm) for 1 min.
7.4.13 Repeat steps 7.4.9 and 7.4.10.
7.4.14 Add 500 µl of Buffer AW1 to each spin column and close the lids.
7.4.15 Centrifuge at 6,000 x g (8,000 rpm) for 1 min.
7.4.16 Repeat steps 7.4.9 and 7.4.10.
7.4.17 Add 500 µl of Buffer AW2 to each spin column and close the lids.
7.4.18 Centrifuge at 20,000 x g (14,000 rpm) for 3 min.
7.4.19 Place each spin column in a 1.5 ml microfuge tube.
7.4.20 Discard previously used collection tubes.
7.4.21 Add 60 µl of Buffer AVE to each spin column.
7.4.22 Close the caps and incubate at room temperature for 1 min.
7.4.23 Centrifuge at 6,000 x g (8,000 rpm) for 1 min.
7.4.24 Discard spin columns and close lids of 1.5 ml microfuge tubes.
7.4.25 Use RNA directly in RT-PCR or store at -80°C until needed.
7.5.1 Screen each sample for HAV RNA and FCV RNA (in two separate reactions, no multiplexing) with one set of Hepatitis A primers(9.1.1) and one set of feline calicivirus primers (9.1.2) using the OneStep RT-PCR kit from Qiagen.
7.5.2 Add 5.0 μl of the sample RNA (7.4.25) to 20.0 μl of RT-PCR reaction mixture (10.5).
7.5.3 Set up a negative control by adding 5.0 µl of RNase-free water to 20.0 µl of the same RT-PCR reaction mixture (10.5). For positive controls, add 5.0 µl of HAV or FCV RNA (as appropriate) that has been previously confirmed as positive in other experiments to 20.0 µl of the same RT-PCR reaction mixture
7.5.4. Insert microfuge tubes in a thermal cycler and proceed with RT-PCR amplification according to the program described under 9.2.
7.5.5 After the RT-PCR is completed, analyze the PCR product by agarose gel electrophoresis (7.6). If necessary, the amplicons can be stored at 4°C until analysis.
7.6.1. Prepare a 2.0% (w/v) agarose gel in 0.5 X TBE (Tris-Borate-EDTA) buffer. The agarose can be dissolved by stirring on a hot plate or by microwaving for 1 to 2 minutes using high power. Ensure that the agarose is completely dissolved (i.e. clear liquid with no particles in suspension).
7.6.2. Cool agarose around 40-50°C and add 2.5 μl of Ethidium Bromide Solution (10mg/mL) or SYBR Safe dye. Gently mix while avoiding bubble formation.
Note 1: The addition of EtBr or SYBR Safe to the gel is optional if the gel is submerged in EtBr or SYBR Safe solution after migration.
Note 2: EtBr IS A POTENT MUTAGEN: USE NITRILE GLOVES WHEN HANDLING.
7.6.3. Pour into a gel tray sealed with tape or as provided by the manufacturer. Avoid bubble formation or bubble trapping. Add a well-forming comb and allow gel to set for about 20 to 30 min.
7.6.4. Prepare samples for electrophoresis: in clean microfuge tubes, mix 2 μl of tracking dye (loading buffer 5x concentrated ) with 10 μl of PCR product.
7.6.5. When the agarose gel has set, remove the comb from the tray, place the tray with gel in the electrophoresis apparatus and fill reservoir with 0.5 X TBE buffer to cover gel with buffer to a depth of 4 mm. Gently pipet approximately 12 μl of samples (7.6.4) into the wells of the submerged gel. Pipet a sample of DNA molecular size marker (e.g., 100 bp ladder DNA) in an empty well. Include positive, negative and reagent controls.
7.6.6. Connect apparatus to power supply with cathode (-, black) situated at the top (i.e., near sample wells) and anode (+, red) at the bottom (i.e., the end) of the gel. Apply approximately 135 volts to gel and run for about 30 minutes or until the tracking dye has spread a distance of approximately two-thirds the length of the gel.
Note : The voltage and time of migration can be changed according to the power supply used.
7.6.7. Remove gel from tray and visualize DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator. Gels may be photographed on Polaroid 667 film to facilitate analysis and for record keeping purposes. Alternatively a digital processing system may be used.
Note 1: In the case where the EtBr has not been added directly to the gel, the gel must be removed from the tray and DNA stained by placing in ethidium bromide (EtBr) solution (10 g/ml) for 10 min. Remove the gel from EtBr using a gel scoop, rinse briefly with tap water, and visualize DNA bands by exposure to UV light.
Note 2: UV LIGHT CAN CAUSE EYE DAMAGE: WEAR SAFETY GOGGLES
7.7.1 The amplicon (PCR product) generated by the HAV primers and the FCV primers are double stranded DNA fragments of 172 bp and 218 bp, respectively. Therefore, a positive PCR test will yield a DNA fragment specific to the targeted gene sequence and will appear as an intense band on an EtBr or SYBR Safe-stained agarose gel. The molecular size of the band can be verified by comparing its migration to that of a DNA molecular size marker (e.g., 100 bp ladder DNA) run on the same gel.
7.7.2 A negative PCR test will normally not produce any visible bands in an EtBr or SYBR Safe-stained agarose gel. Although an extremely rare occurrence, any sample giving bands not corresponding to the expected amplicon (non-specific amplification products) is considered to be negative.
7.7.3 A specific band should appear for the targeted positive control. Absence of a positive control band invalidates the test and the samples should be re-analyzed.
7.7.4 Any band corresponding to the positive control occurring in the negative or reagent control indicates contamination problems with the PCR reaction mixture and the whole batch is considered suspect and should be discarded. The samples should be re-analysed using a new reagent batch
Amplicons taken from the presumptive positives should be sent to a reference laboratory for sequencing, along with the food extracts (if available) for confirmation of the analysis. The reader is invited to contact the authors listed above.
7.7.5 Any test sample showing a distinct band, corresponding to its positive control, is considered as a presumptive positive. The PCR product should be confirmed by sequence analysis.
8.1 QIAamp® Viral RNA Mini kit insert, printed January 1999.
8.2 Qiagen OneStep® RT-PCR kit insert, printed May 2002
8.3 Matrix Microscience Inc. Accessed on July 17th, 2006 at http://www.matrixmsci.com/
8.4 Papafragkou, E., Plante, M., Mattison, K., Bidawid, S., Karthikeyan, K., Farber, J. M., and Jaykus, L.-A. 2007. Rapid and sensitive detection of hepatitis A virus in representative food matrices. J. Virol. Meth.147:177-87.
8.5 Guévremont, E., Brassard, J., Houde, A., Simard, C., and Trottier, Y.-L. 2006. Development of an extraction and concentration procedure for the detection of hepatitis A virus and norovirus from green onions by RT-PCR. J. Virol. Meth. 134: 130-5.
9.1.1 Screening RT-PCR for HAV: Oligonucleotide primers for HAV are based on the protease coding region 2 A of HAV and give an amplification product iof 172 bp (8.5).
Guévremont primers for the detection of HAV (172 bp fragment) :
SH-Prot-A-forward - 20 bases (3378-3397) = 5'-ATG GAT GCT GGR GTT CTT AC-3'
SH-Prot-1- reverse - 22 bases (3529-3550 = 5'-ART TGG CAG CAA TTT CTT CAA G-3'
Note: Numbers refer to the corresponding nucleotide positions of reference hepatitis A virus strain HM-175 wild type (GenBank accession number NC 001489
|R||A, G||H||A, C, T|
|Y||C, T||B||C, G, T|
|M||A, C||V||A, C, G|
|K||G, T||D||A, G, T|
|S||C, G||N||A, C, G, T|
|W||A, T||X||A, C, G, T|
9.2.1 Screening RT-PCR for FCV. FCV primers were designed in-house by Dr. César Bin Kingombe, Health Canada.
FCV primers for the detection of feline calicivirus (218 bp fragment) :
CBK-1 - upstream 20 bases = 5'-GGA GGC GCG ATC TTC AGT AT- 3'
CBK-2 - downstream 20 bases = 5'-GCA TAA CTC GTC GGA GGT GT-3'
Note: Synthesis of oligonucleotideprimers can usually be contracted out to a local university or, alternatively,many biotechnology firms offer a custom synthesis service. If assistance is required in this matter, contact the authors.
The thermal cycler program should be set for the following sequence of cycling parameters :
|1||Reverse transcription||30 minutes||50°C|
|2||Initial PCR activation step||15 minutes||95°C||HotStarTaq DNA Polymerase)Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA
template is denatured
|3||40 cycles of
(3.1+ 3.2+ 3.3)
|4||Final elongation||10 minutes||72°C|
Note: The use of thermal cyclers other than the models stated above may alter the performance of the PCR, and it may be necessary, for the user, to optimize cycling parameters for different models.
15.01 g Glycine (molecular biology grade).
32.73 g NaCl (molecular biology grade).
8 ml Tween 20 (molecular biology grade).
Add reagents to 3.5 L of distilled water, dissolve reagents with a magnetic stirrer and adjust the pH to 9.0. Complete to 4.0 L and filter sterilize.
Note: The glycine buffer pH 9.0 is stable for 9 months at 4 °C.
All stock solutions are stored at -20°C until use. The following is a recipe for preparing a large batch equivalent to 20 reactions.
Note : All reagents, Dnase RNase-free water, pipet tips and other materials coming into contact with samples or RT-PCR reagents should be sterile or autoclaved prior to use to remove any DNAses and/or other contaminants. To avoid contamination problems, all reagents should be prepared in a laminar flow cabinet which has never been exposed to Hepatitis A virus or Hepatitis A virus PCR products. To avoid any non-specific amplifications, the mix should be prepared by putting all the reagents on ice or on a refrigerated rack. Also, for reducing the cost of reagents, 20 μl of RT-PCR mix in 200 μl tubes are used.
|RT-PCR Components||Initial concentration||Stock solutions required for 20 reactions tubes using HAV primers||Volume
|RNase-free water||200.0 μl||10 μl|
|5x QIAGEN OneStep RT-PCR Buffer||5x||100.0 μl||5 μl||1 x|
|dNTP Mix||10 mM∕of each dNTP||20.0 μl||1 μl||400 μM|
|10 μM∕per primer|| 30.0 μl
||1.5 μl||0.6 μM|
|10 μM∕per primer||30.0 μl||1.5 μl||0.6 μM|
|QIAGEN OneStep RT-PCR Enzyme Mix||20.0 μl||1 μl|
|Total Volume||400 μl||20 μl|
|Distribute per tube||20 μl|
Tris Base (molecular biology grade). 54.0 g
Boric Acid (molecular biology grade). 27.5 g
EDTA disodium (molecular biology grade). 3.75 g
Add distilled water to a volume of 800 mL, dissolve, complete to 1.0 L.
This buffer is used at a 1:10 dilution (TBE 0.5 X Buffer) in distilled water.
The pH of the 0.5 X buffer should be 8.3. Do not adjust the pH.
10X Orange G, sodium salt 0.025 g 0.25% (w/v)
Glycerol 4 mL 40% (w/v)
Sterile distilled water (DNAse RNAse Free) 6 mL
Mix all the ingredients thoroughly, sterilize by autoclaving at 121°C for 15 minutes or filter sterilize and store in 1.0 mL aliquots at -20°C.
Note : The tracking dye / loading buffer binding buffer is stable for 12 months at -20°C.
Note : Store the working solution at 4°C.
Although many types of DNA size marker preparations are available from different suppliers, the 100 bp ladder DNA marker provides a useful range of DNA fragment sizes and facilitates the "sizing" of PCR amplicons generated in this reaction.