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OPFLP-07 Detection of Hepatitis A virus using the conventional and real-time reverse-transcriptase polymerase chain reaction

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关键词：: OPFLP-07 Detection of Hepatitis

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内容简介：: Government of Canada Gouvernement du Canada Laboratory Procedure OPFLP-07 March 2010 HEALTH PRODUCTS AND FOOD BRANCH OTTAWA DETECTION OF HEPATITIS A VIRUS USING THE CONVENTIONAL AND REAL-TIME REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION Alain Houde 1, Elyse Poitras 1, Danielle Leblanc 1, Pierre Ward 1, Evelyne Guvremont 1, Julie Brassard 1, Yvon-Louis Trottier 2 and Carole Simard 3 1 Health Canada,Bureau of Microbial Hazards,251 Sir Frederick Banting Driveway,Ottawa,ON, Canada, K1A 0K9 2 Agriculture and Agri-Food Canada,Food Research and Development Centre, 3600 Casavant Blvd.West, St. Hyacinthe, QC, Canada, J2S 8E3 3 St-Hyacinthe Laboratory, Food Virology Unit, Canadian Food Inspection, 3400 Casavant Blvd. West, St. Hyacinthe, QC, Canada, J2S 8E3 Microbiological Methods Committee Evaluation Division Bureau of Microbial Hazards, Food Directorate, Postal Locator: 2204E HPFB, Ottawa, Ontario, K1A 0K9 E-mail : mitteehc-sc.gc.ca 1. APPLICATION This method is applicable to the amplification and detection of hepatitis A virus (HAV) RNA by conventional and real-time (TaqMan) reverse-transcriptase polymerase chain reaction (RT-PCR) following the extraction of nucleic acids from a clinical, environmental or food matrix. This method will not differentiate between infectious and non-infectious viruses. 2. DESCRIPTION This protocol is routinely used to amplify and detect HAV RNA isolated from strawberries, green onions and water (please see OPFLP-09, OPFLP-03 and OPFLP-04). 3. PRINCIPLE Total RNA is extracted from a food, clinical or environmental sample using an appropriate procedure or a cell free sample (for instance, HAV stock suspension) using the QIAamp viral RNA Mini Kit (Qiagen). The final RNA extract is used for conventional and/or real-time RT-PCR procedure which amplifies a specific fragment depending of the primers used. HAV amplified fragments are confirmed by sequencing after gel electrophoresis by conventional RT-PCR or by fluorescence reading of the TaqMan probe degradation by the Taq polymerase exonuclease activity during the amplification process in real-time RT-PCR. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 2 - 4. DEFINITION OF TERMS 4.1 See Appendix A of Volume 3. 5. COLLECTION OF SAMPLES 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). Hepatitis A virus could also have been previously propagated in FRhK-4 cells, aliquoted in 2-ml cryogenic vials and stored at -70C. SAFETY NOTE: TAKE APPROPRIATE LABORATORY MEASURES WHEN HANDLING HAV. CONTAINMENT REQUIREMENTS: BIOSAFETY LEVEL 2 PRACTICES AND CONTAINMENT FOR ACTIVITIES WITH INFECTED MATERIALS. PROTECTIVE CLOTHING: LABORATORY COAT; GLOVES WHEN DIRECT CONTACT WITH INFECTIOUS MATERIALS IS UNAVOIDABLE; SLEEVE PROTECTORS AND GLOVES (NITRILE) FOR WORK IN BIOSAFETY CABINET. IMMUNIZATION: IMMUNIZATION (HAV VACCINE) IS RECOMMENDED FOR EMPLOYEES HANDLING HAV. FOR MORE INFORMATION CONSULT THE PUBLIC HEALTH AGENCY OF CANADA LEVEL 2 BIOSAFETY GUIDELINES AT http:/www.phac-aspc.gc.ca/dpg_e.html#biosafety AND APPROPRIATE MSDSS AT http:/www.phac-aspc.gc.ca/msds-ftss/msds75e.html 6. MATERIALS AND SPECIAL EQUIPMENT Note: 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 17025:2005 (or latest version): General requirements for the competence of testing and calibration laboratories. 6.1 Thermal cycler (Eppendorf Mastercycler Gradient or equivalent). 6.2 Spectrofluorometric thermal cycler (Stratagene Mx series or equivalent). 6.3 Microwave oven or hot plate. 6.4 Submarine gel casting tray and buffer reservoir, power pack and 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 with photographic filters for ethidium bromide and/or SYBR Safe DNA gel stain. 6.7 Adjustable micropipettors to cover range of volumes: 0.5-10 l, 10-100 l, and 100-1000 l with filtered pipet tips. 6.8 Microcentrifuge for microfuge tubes -1.5 ml/2.0 ml capacity and a speed range up to 20000 x g (Labnet International Spectrafuge 16 M or equivalent). OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 3 - 6.9 Standard heating blocks (VWR scientific products or equivalent) or waterbath capable of accommodating 1.5 ml microfuge tubes and capable of maintaining a temperature of 37-50C. Note: It is the responsibility of each laboratory to ensure that the block heaters or water baths are maintained at the recommended temperatures. Where 37C is recommended the waterbath may be at 37C +/- 1C. For all other temperatures it may be +/- 2C. 6.10 Vortex mixer. 6.11 Sterile microfuge tubes - 2.0 ml, 1.5 ml capacity and/or 96-well plate. 6.12 Tubes for PCR - thin wall 0.2 ml or 0.5 ml capacity, strip or 96-well plate (depending on thermal cycler model). 6.13 Timer. 6.14 Container for ice or cooling rack. 6.15 Freezers capable of maintaining -20C and -70C. 6.16 QIAamp viral RNA extraction kit (Qiagen product number 52904 or 52906, or equivalent). 6.17 Qiagen OneStep RT-PCR kit (Qiagen product number 210210 or 210212, or equivalent). 6.18 Stratagene Brilliant II QRT-PCR Core Reagent kit, 1-Step (Stratagene product number 600810 or equivalent). 6.19 Non-denatured ethanol 96 - 99% (room temperature 23 C +/- 3 C). 6.20 HAV primers (SH-Prot-A and SH-Prot-1; SH-Poly-A and SH-Poly-1) for the detection of HAV by conventional RT-PCR (See section 9.1 for DNA sequences). 6.21 HAV primers (SH-Prot-A and SH-Prot-1; SH-Poly-A and SH-Poly-1) and TaqMan probes (SH-Prot-Q; SH-Poly-Q) for the detection of HAV by real-time RT-PCR (See section 10.1 for DNA sequences). 6.22 QIAquick gel extraction kit (Qiagen product number 28704 or 28706, or equivalent). 6.23 TOPO TA Cloning kit (with pCR2.1-TOPO vector) with One Shot TOP10 Electrocomp E. coli (Invitrogen product number K4560-01 or K4560-40, or equivalent). 6.24 X-GAL (5-Bromo-4-Chloro-3-Indolyl-D-Galactopyranoside) (BioShop product number XGA001 or equivalent). 6.25 Electroporation system (Bio-Rad Gene Pulser product or equivalent). 6.26 Electroporation cuvettes Gene Pulser/MicroPulser cuvettes, 0.1 cm gap, (Bio-Rad product number 165-2089 or equivalent). 6.27 Kanamycin, 10 mg/ml (Invitrogen product number 18161-054 or equivalent). 6.28 LB broth Miller (BioShop product number LBL407-50 or equivalent). OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 4 - 6.29 NucleoSpin Plasmid kit (Macherey Nagel product number MCN540788050 or MCN540788250, or equivalent). 6.30 EcoRI restriction endonuclease and buffer (Invitrogen product number 15202-021, or equivalent). 6.31 BamHI restriction endonuclease and buffer (Invitrogen product number 15201-031, or equivalent). 6.32 Spectrophotometer (Thermo Scientific NanoDrop 1000 or equivalent). 6.33 In addition, the following chemicals and reagents should be on hand. Dimethylformamide (DMF), reagent grade (BioShop product number DMF451 or equivalent). Agar (Laboratory grade). 15 ml snap cap tubes (e.g., Falcon). Agarose (molecular biology grade). DNA Ladder 100 bp (or equivalent). Ethidium bromide (molecular biology grade) or SYBR Safe DNA gel stain (Invitrogen product number S33102 or equivalent). Nucleic acid sample loading buffer (10X) (BlueJuiceTM Gel Loading Buffer, Invitrogen product number 10816-015 or equivalent). RNase inhibitor (RNaseOUTTM Recombinant Ribonuclease Inhibitor 40 U/l, Invitrogen product number 10777-019 or equivalent). 0.5X TBE (Tris-Borate-EDTA) or 1X TAE (Tris-Acetate-EDTA) buffer. RNase-free water (molecular biology grade). Salmon sperm DNA solution 10 mg/ml (Invitrogen product number 15632-011 or equivalent. 7. PROCEDURE 7.1 Viral RNA extraction from cell free samples using the QIAamp Viral RNA Mini kit Note 1: All reagents used in this section are provided in the kit with the exception of ethanol 96-99% and RNase inhibitor. The protocol for nucleic acid extraction from viruses has been established according to the manufacturers instructions provided in the kit manual. AVL buffer must be completely dissolved prior to being used. Always follow the latest manufacturers instructions provided in the QIAamp Viral RNA Mini kit. The most up-to-date manufacturers instructions will prevail over the herein instructions. Note 2: Great care should be taken when working with RNA to avoid contact with ubiquitous Rnases. Rnases are very stable and are difficult to inactivate. Always store the extracted RNA with 20 units of Rnase inhibitor and wear gloves while handling reagents and samples. Use sterile, disposable Rnase-free plasticware. Glassware should be treated (cleaned and thoroughly rinsed and baked at 240C for four hours or more before use). 7.1.1 Pipet 560 l of prepared buffer AVL containing carrier RNA into a 1.5 ml microfuge tube. Note: Buffer AVL-carrier RNA should be prepared fresh, and is stable at 2 8 C for up to 48 hours. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 5 - 7.1.2 Add 140 l of each sample into a microfuge tube containing the buffer AVL-carrier RNA. SAFETY NOTE: BUFFERS AVL AND AW1 CONTAIN CHAOTROPIC SALT WHICH IS AN IRRITANT. TAKE APPROPRIATE LABORATORY MEASURES AND WEAR GLOVES WHEN HANDLING. THEY ARE NOT COMPATIBLE WITH DISINFECTING AGENTS THAT CONTAIN BLEACH. DISPOSE OF ALL SOLUTIONS, BUFFERS AND REAGENTS ACCORDING TO THE WASTE-DISPOSAL GUIDELINES. 7.1.3 Pulse-vortex microfuge tubes for 15 seconds. 7.1.4 Incubate the samples for 10 minutes at room temperature (23C +/- 3 C). 7.1.5 Briefly centrifuge the microfuge tubes to remove drops from the inside of the lid. Note: Each centrifugation step is carried out at room temperature (23 C +/- 3 C). 7.1.6 Add 560 l of non-denatured ethanol (96 - 99%) to the samples. 7.1.7 Pulse-vortex microfuge tubes for 15 seconds. 7.1.8 Transfer 630 l of the solution to a QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim. 7.1.9 Close the caps and centrifuge the spin columns at 6000 x g for 1 minute. 7.1.10 Transfer each spin column to a clean 2-ml collection tube. 7.1.11 Discard the previously used collection tube. 7.1.12 Transfer the remaining 630 l of each solution to its respective spin column. 7.1.13 Centrifuge at 6000 x g for 1 minute. 7.1.14 Repeat steps 7.1.10 and 7.1.11. 7.1.15 Add 500 l of buffer AW1 to each spin column and close the lids. 7.1.16 Centrifuge at 6000 x g for 1 minute. 7.1.17 Transfer each spin column to a clean 2 ml collection tube. 7.1.18 Discard previously used collection tube. 7.1.19 Add 500 l of buffer AW2 to each spin column and close the lids. SAFETY NOTE: BUFFERS AW2 AND AVE CONTAIN SODIUM AZIDE AS PRESERVATIVE WHICH IS HIGHLY TOXIC. TAKE APPROPRIATE SAFETY MEASURES AND WEAR GLOVES WHEN HANDLING. DISPOSE OF ALL SOLUTIONS, BUFFERS AND REAGENTS ACCORDING TO THE WASTE-DISPOSAL GUIDELINES. 7.1.20 Centrifuge at 20000 x g for 3 minutes. 7.1.21 Place each spin column in a 1.5 ml microfuge tube. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 6 - 7.1.22 Discard previously used collection tube. 7.1.23 Add 60 l of buffer AVE to each spin column. Note: Alternatively, the addition of 2 X 30 l of buffer AVE (double elution) can be performed. (i.e., two separate applications of buffer AVE to each QIAamp Mini spin columns followed by separate centrifugation steps at 6000 x g for 1minute) (8.5). 7.1.24 Close the caps and incubate at room temperature (23C +/- 3 C) for 1 minute. 7.1.25 Centrifuge at 6000 x g for 1 minute. 7.1.26 Discard spin columns and close lids of 1.5 ml microfuge tubes. 7.1.27 Add 20 units of RNase inhibitor (0.5 l of RNaseOUTTM 40 U/l) to the extracted RNA. 7.1.28 Use RNA directly in RT-PCR, real-time RT-PCR or store at -70C until needed. 7.2 Handling of RNA extracted from sample units 7.2.1 During storage and transport keep the extracted RNA from sample units frozen (-70C). 7.2.2 Extracted RNA should be kept on ice while preparing the conventional or the real-time RT-PCR reactions. 7.3 Preparation for analysis 7.3.1 Ensure that all reagents needed are available. 7.3.2 Prepare and verify the suitability of all controls. Include a negative and a positive amplification control for both RT-PCR tests (conventional or real-time) that are specific to the primers used. Negative amplification controls use water as template to ensure there has been no contamination of the PCR mix. Positive amplification controls use purified HAV RNA that has been previously confirmed as positive in other experiments or corresponding cDNA clone (11.7) to ensure the PCR reagents and primers have the intended specificity. 7.3.3 HAV titre can be estimated using conventional RT-PCR. The titre will then be expressed in RT-PCR units. Thaw a 2 ml cryogenic vial containing a reference HAV strain (for instance, HM175) RNA on ice. Once thawed, make 10-fold dilution series of the virus by adding 100 l of virus stock to 900 l of RNase-free water in a 1.5 ml microfuge tube (this is the 10-1 dilution). Repeat this step by taking 100 l of the 10-1 viral dilution and add to 900 l of RNase-free water in another 1.5 ml microfuge tube (this is now the 10-2 dilution). Repeat the previous step in order to reach the 10-8 dilution. 7.3.4 Standard curve for real-time RT-PCR systems is generated in triplicate using 10-fold serial dilutions (108 to 100 genomic equivalents) of purified linear cDNA plasmid (11.6 and 11.7) containing the PCR product obtained with each primer set system respectively in a 5 ng/ml salmon sperm DNA solution. Once thawed, make 10-fold dilution series of the cDNA plasmid by adding 10 l of plasmid stock to 90 l of RNase-free water containing salmon sperm DNA (5 ng/ml) in a 1.5 ml microfuge tube (this is the 10-1 dilution). Repeat this step by taking 10 l of the 10-1 cDNA plasmid dilution and add to 90 l of RNase-free water containing salmon OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 7 - sperm DNA (5 ng/ml) in another 1.5 ml microfuge tube (this is now the 10-2 dilution). Repeat the previous step in order to reach the 10-8 dilution. 7.4 Conventional RT-PCR method for the amplification of HAV RNA 7.4.1 Amplify RNA from each extracted RNA sample with the set of HAV primers (SH-Prot-A and SH-Prot-1 and/or SH-Poly-A and SH-Poly-1) using the OneStep RT-PCR kit from Qiagen. 7.4.2 Add 5.0 l of each extracted RNA sample to 20.0 l of RT-PCR reaction mixture (9.3). 7.4.3. Set up a negative control by adding 5.0 l of RNase-free water to 20.0 l of the same RT-PCR reaction mixture (9.3). For positive controls, add 5.0 l of HAV RNA to 20.0 l of the same RT-PCR reaction mixture. 7.4.4 Insert PCR tubes or 96-well plate in a thermal cycler and proceed with RT-PCR amplification according to the program described under 9.2. 7.4.5 After the RT-PCR is completed, analyze the RT-PCR product by agarose gel electrophoresis (7.5). If necessary, the amplicons can be stored at 4C until analysis. 7.5 Agarose gel electrophoresis 7.5.1. Prepare a 2.0% (w/v) agarose gel in 0.5 X TBE (Tris-Borate-EDTA) or 1 X TAE (Tris-Acetate-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). 7.5.2. Cool agarose to around 45C then add the concentrated ethidium bromide (EtBr) solution to obtain a final concentration of 0.5 g/ml in the agarose gel. Gently mix while avoiding bubble formation. Note 1: Alternatively, SYBR Safe DNA gel stain can be used instead of EtBr. Dilute the concentrated stain to have a final dilution of 1:10000 in agarose gel (8.8). Note 2: The addition of EtBr/ SYBR Safe DNA gel stain to the gel is optional if the gel is submerged either in EtBr or SYBR Safe DNA gel stain solution after migration. Note 3: Select the appropriate photographic filter when using either EtBr or SYBR Safe DNA gel stain. SAFETY NOTE: EtBr IS A POTENT MUTAGEN: USE NITRILE GLOVES WHEN HANDLING. DISPOSE OF ALL SOLUTIONS AND USED GELS ACCORDING TO THE WASTE-DISPOSAL GUIDELINES. 7.5.3. Pour into a gel tray. Avoid bubble formation or bubble trapping. Add a well-forming comb and allow gel to solidify for about 20 to 30 minutes. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 8 - 7.5.4. Prepare samples for electrophoresis: in clean microfuge tubes, mix 1.2 l of tracking dye (nucleic acid loading buffer 10 X concentrated) into each microfuge tubes containing 10 l of RT-PCR product. 7.5.5. When the agarose gel has solidified, remove the comb and place the tray with gel in the electrophoresis apparatus and fill reservoir with 0.5 X TBE or 1 X TAE buffer to cover gel with buffer to a depth of 4 mm. Gently pipet the samples (approximately 11.2 l) (7.5.4) for electrophoresis into the wells of the submerged gel. Include a DNA molecular size marker (e.g., 100 bp DNA ladder), as well as positive, negative and reagent controls. 7.5.6. 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 100 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 modified according to the size of the electrophoresis device (distance between electrodes) and the length of the gel. 7.5.7 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: In the case where the EtBr / SYBR Safe has not been added directly to the gel, the gel must be removed from the tray and DNA stained by placing the gel in ethidium bromide (EtBr) solution (10 g/ml) or SYBR Safe 1 X solution for 15 minutes. Remove the gel from EtBr or SYBR Safe solution using a gel scoop, rinse briefly with tap water, and visualize DNA bands by exposure to UV light. SAFETY NOTE: UV LIGHT CAN CAUSE EYE DAMAGE: WEAR SAFETY GOGGLES. 7.6 Reading conventional RT-PCR results 7.6.1 The amplicons (RT-PCR products) generated by the HAV SH-Poly primers and SH-Prot primers are double stranded DNA fragments of 107 bp and 172 bp, respectively. Therefore, a positive PCR test will yield a DNA fragment specific to the targeted gene sequence and will appear as an intense DNA band on an EtBr / 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 DNA ladder) run on the same gel. 7.6.2 A negative PCR test will normally not produce any visible DNA bands in an EtBr / SYBR Safe stained agarose gel. Although in an extremely rare occurrence, any sample giving bands not corresponding to the expected amplicon (non-specific amplification products) is considered to be negative. 7.6.3 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-amplified. 7.6.4 Any band corresponding to the positive control occurring in the negative control with the HAV primers indicates that sample contamination may have occurred with the RT-PCR reaction mixture and the whole batch is considered suspect and should be discarded. The samples should be re-amplified using a new reagent batch. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 9 - 7.6.5 Any test sample showing a distinct band with the HAV primers, corresponding to its positive control, is considered as a presumptive positive. For field (food, clinical or environmental) samples, the RT-PCR product must be confirmed as a HAV strain by sequencing. Note: For food samples, it is recommended to send the extracted RNA obtained from positive samples to the Bureau of Microbial Hazards of Health Canada for further molecular characterization of the HAV strain. 7.7 Calculating RT-PCR units 7.7.1 After reading the RT-PCR results (7.6), determine the highest HAV dilution (7.3.3) showing a positive result (7.6.5) which is called the end-point dilution. 7.7.2 Once determined, take the reciprocal of this dilution to give RT-PCR units per quantity used for the RT-PCR (i.e., 5.0 l). 7.7.3 To obtain a concentration per ml, multiply the concentration obtained in step 7.7.2 by a factor that will give you 1000 l or 1 ml (i.e., if 5.0 l of RNA were used for the RT-PCR and 10-6 was the highest dilution in which a band was observed, multiply 106 by 200). Therefore, the RT-PCR units are 2.0 x 108 RT-PCR units per ml. Note: Viral RNA titres are usually expressed as RT-PCR units per ml. 7.8 Real-time RT-PCR method for the amplification of HAV RNA 7.8.1 Amplify RNA from each extracted RNA sample in duplicate with the set of HAV primers and TaqMan probes (SH-Prot-A, SH-Prot-1 and SH-Prot-Q; and/or SH-Poly-A, SH-Poly-1 and SH-Poly-Q) using the Brilliant II QRT-PCR Core Reagent kit from Stratagene or equivalent. Perform, in triplicate, a minimum of 5 to 6 points of the corresponding validated standard curve of known concentration of copy numbers. 7.8.2 Add 2.0 l of each extracted RNA sample or clone used for the standard curve to 23.0 l of real-time RT-PCR reaction mixture (10.3). 7.8.3. Set up reaction controls in triplicate. For the No Template Control (NTC), add 2.0 l of RNase-free water to 23.0 l of the same real-time RT-PCR reaction mixture (10.3). As positive control, add 2.0 l of HAV RNA that has been previously confirmed as positive in other experiments or cDNA from the corresponding clone to 23.0 l to the same real-time RT-PCR reaction mixture. In the case of a two-step RT-PCR reaction, a no-RT control reaction could be included by omitting the reverse transcriptase enzyme in the RT reaction. The no-RT control is expected to generate no real-time PCR signal using specific primers and probe with the previously confirmed HAV genomic RNA samples. 7.8.4 Insert PCR strip tubes or 96-well plate in the spectrofluorometric thermal cycler and proceed with RT-PCR amplification according to the program described under 10.2. Fluorescence readings should be taken at the annealing/extension temperature (60C). 7.8.5 When the real-time RT-PCR run is completed, analyze the reactions using the MX series software or the software provided with the spectrofluorometric thermal cycler. If necessary, the amplicons can be stored at 4C for further analysis. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 10 - 7.9 Reading real-time RT-PCR results 7.9.1 Results are displayed in an amplification plot, which reflects the change in fluorescence during cycling. A positive real-time RT-PCR test will generate a Ct (threshold cycle) that is derived from either raw fluorescence or normalized fluorescence (with reference dye, for instance Rox). As PCR proceeds, the fluorophore will produce fluorescent light (degradation of the TaqMan probe) of a color that is characteristic of the fluorophore used over the background fluorescence of the linear probe quench. The Ct is defined as the cycle at which the fluorescence is determined to be statistically significant above the background signal contributed by the fluorescence-labelled oligonucleotide within the PCR reaction. Check for any abnormalities in the amplification plot. 7.9.2 If a standard curve is used, the expected concentration range of the sample should fall within the concentration range of the standard curve. Ideally Ct values would fall between 15 and 35. A plot of Ct vs the logarithm of the copy number corresponding to that Ct results in a straight line. An efficient real time PCR assay has a slope of -3.3, an intercept of around 38 and a R2 greater than 0.98. The efficiency of the amplification should be between 90 and 110% (optimal standard curves are based on amplification efficiencies of as close to 100% as possible). 7.9.3 A negative real-time RT-PCR test will normally not produce any fluorescence over the background signal of the probe for the entire run. It will be validated with the positive control. 7.9.4 At a specific previously validated Ct, the fluorescence from positive control should increase over the background fluorescence of the linear probe quench. Absence of fluorescence in the positive control invalidates the test and samples should be re-amplified. 7.9.5 Any Ct occurring in the negative control with the HAV primers and probe set indicates that sample contamination may have occurred with the real-time RT-PCR reaction mixture and the whole batch is considered suspect and should be discarded. The samples should be re-amplified using a new reagent batch. 7.9.6 Any sample showing a Ct with the HAV primers and probe set when NTC are negative or when NTC Ct 5 cycles from the highest Ct value of the samples analyzed is considered as positive. Note: For food samples, it is recommended to send the extracted RNA obtained from positive samples to the Bureau of Microbial Hazards of Health Canada for further molecular characterization of the HAV strain. 7.10 Calculating genomic equivalent units 7.10.1 Unknown sample Ct value must fall on the standard curve to be quantified in genomic equivalent/volume. Initial genomic equivalent values of cDNA can be estimated based on the mean of the duplicate threshold cycle (Ct) of the sample analyzed compared to its respective (HAV Poly or Prot) standard curve. 7.10.2 To obtain the initial concentration in genomic equivalents per l, divide the concentration obtained in step 7.10.1 by the volume of RNA used as template (i.e., if 2.0 l of RNA were used for the real-time RT-PCR reaction and the mean Ct value obtained corresponds to 4.0 x 106 genomic equivalents on the standard curve, divide 4.0 x 106 genomic equivalents by 2 l). Therefore, the concentration is 2.0 x 106 genomic equivalents per l). OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 11 - Note: Viral RNA concentrations are usually expressed as genomic equivalent per l of RNA used as template. By using appropriate calculations, it could be expressed as genomic equivalent per unit of sample tested. 8. REFERENCES 8.1 Brilliant II QRT-PCR Core Reagent Kit, 1-Step Instruction Manual. 2006. Available at: /manuals/600810.pdf 8.2 Guvremont E., Brassard J., Houde A., Simard C., Trottier Y.-L. 2006. Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions. Journal of Virological Methods 134: 130-135. 8.3 Houde A., Guvremont E., Poitras E., Leblanc D., Ward P., Simard C., Trottier Y.-L. 2007. Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus. Journal of Virological Methods 140: 80-89. 8.4 NucleoSpin Plasmid kits user Manual. March 2008. Available at: http:/www.mn-/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf 8.5 QIAamp Viral RNA Mini Handbook. December 2007. Available at: /literature/handbooks/literature.aspx?id=1000199 8.6 Qiagen OneStep RT-PCR Kit Handbook. February 2008. Available at: /literature/handbooks/literature.aspx?id=1000223 8.7 QIAquick Spin Handbook. March 2008. pp. 25-26. Available at: /literature/handbooks/literature.aspx?id=1000252 8.8 SYBR Safe DNA gel stain. July 2006. Available at: /media/pis/mp33100.pdf 8.9 TOPO TA Cloning user Manual. April 2006. Available at: /content/sfs/manuals/topota_man.pdf 9. CONVENTIONAL RT-PCR REAGENTS AND TEMPERATURE CYCLING PROGRAM 9.1 RT-PCR primers SH-Poly primers (8.2) for the detection of HAV (107 bp fragment): SH-Poly-A (forward) 26 bases = 5 - GAR TTT ACT CAG TGT TCA ATG AAT GT - 3 SH-Poly-1 (reverse) 20 bases = 5 - GGC ATA GCT GCA GGA AAA TT -3 SH-Prot primers (8.2) for the detection of HAV (172 bp fragment): OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 12 - SH-Prot-A (forward) 20 bases = 5 - ATG GAT GCT GGR GTT CTT AC - 3 SH-Prot-1 (reverse) 22 bases = 5 - ART TGG CAG CAA TTT CTT CAA G -3 Standard MixBase Definitions Letter Nucleotides Letter Nucleotides 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 Note: Synthesis of oligonucleotide primers 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. 9.2 Temperature cycling program for HAV primers Using the SH-Poly primer system, the thermal cycler program should be set for the following sequence of cycling parameters: Step 1 Reverse transcription 30 minutes 50 C Step 2 Initial PCR activation step 15 minutes 95 C HotStarTaq DNA Polymerase is activated; Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA template is denatured (8.6). Step 3 40 cycles of: Step 3.1 Denaturation 45 seconds 94 C Step 3.2 Annealing 45 seconds 50 C Step 3.3 Extension 45 seconds 72 C Step 4 Final elongation 10 minutes 72 C Note 1: The use of thermal cyclers other than the models stated above or the use of other RT-PCR reagent kit than the Qiagen OneStep RT-PCR kit may alter the performance of the RT-PCR reactions. It may be necessary, for the user, to optimize cycling parameters for different models or reagent kit. Note 2: For better results, it is recommended to use primers that have been purified by High Pressure/Performance Liquid Chromatography (HPLC). OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 13 - Using the SH-Prot primer system, the thermal cycler program should be set for the following sequence of cycling parameters: Step 1 Reverse transcription 30 minutes 50 C Step 2 Initial PCR activation step 15 minutes 95 C HotStarTaq DNA Polymerase is activated; Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA template is denatured (8.6). Step 3 40 cycles of: Step 3.1 Denaturation 45 seconds 94 C Step 3.2 Annealing 45 seconds 53 C Step 3.3 Extension 45 seconds 72 C Step 4 Final elongation 10 minutes 72 C Note 1: The use of thermal cyclers other than the models stated above or the use of other RT-PCR reagent kit than the Qiagen OneStep RT-PCR kit may alter the performance of the RT-PCR reactions. It may be necessary, for the user, to optimize cycling parameters for different models or reagent kit. Note 2: For better results, it is recommended to use primers that have been purified by High Pressure/Performance Liquid Chromatography (HPLC). OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 14 - 9.3 Qiagen OneStep RT-PCR kit (8.6) All stock solutions are stored at -20C until used. The following is a recipe for preparing a large batch equivalent to 20 reactions. Note 1: The protocol for RT-PCR amplification has been established according to the manufacturers instructions provided in the kit manual. Always follow the latest manufacturers instructions provided in Qiagen OneStep RT-PCR kit. The most up-to-date manufacturers instructions will prevail over the herein instructions. Note 2: Great care should be taken when working with RNA to avoid contact with ubiquitous RNases. RNases are very stable and are difficult to inactivate. Always store the extracted RNA with 20 units of RNase inhibitor and wear gloves while handling reagents and samples. Use sterile, disposable RNase-free plasticware. Glassware should be treated (cleaned and thoroughly rinsed and baked at 240C for four or more hours before use). Note 3: 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 HAV or HAV RT-PCR products. To avoid any non-specific amplification, the mix should be prepared by putting all the reagents on ice or on a refrigerated rack. Also, for reducing the cost of reagents, 25 l of RT-PCR mix in 200 l tubes are used. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 15 - RT-PCR Components Initial concentration Stock solutions required for 20 reactions tubes using SH-primers Volume per tube Final Concentration RNase-free water - 200.0 l 10.0 l - 5 X QIAGEN OneStep RT-PCR buffer 5 X 100.0 l 5.0 l 1 X dNTP Mix 10 mM of each dNTP 20.0 l 1.0 l 400 M SH-Prot-A or SH-Poly-A primer 10 M 30.0 l 1.5 l 0.6 M SH-Prot-1 or SH-Poly-1 primer 10 M 30.0 l 1.5 l 0.6 M QIAGEN OneStep RT-PCR Enzyme Mix - 20.0 l 1.0 l - Total Volume 400.0 l 20.0 l - Distribute per tube 20.0 l - - Template per tube - 5.0 l - OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 16 - 10. REAL-TIME RT-PCR REAGENTS AND TEMPERATURE CYCLING PROGRAM 10.1 Real-time RT-PCR primers and probes SH-Poly primers (8.3) for the detection of HAV using a TaqMan assay (107 bp fragment): SH-Poly-A (forward) 26 bases = 5 - GAR TTT ACT CAG TGT TCA ATG AAT GT - 3 SH-Poly-1 (reverse) 20 bases = 5 - GGC ATA GCT GCA GGA AAA TT -3 SH-Poly probe (8.3) for the detection of HAV using a TaqMan assay: SH-Poly-Q 28 bases = 5 TCT CCA AAA CGC TTT TTA GAA AGA GTC C - 3 Note 1: Synthesis of oligonucleotide primers can usually be contracted out to a local university or, alternatively, many biotechnology firms offer a custom synthesis service. It is advisable to order oligonucleotides that have been purified by High Pressure/Performance Liquid Chromatography (HPLC) for real-time RT-PCR applications. If assistance is required in this matter, contact the authors. Note 2: SH-Poly-Q TaqMan probe was ordered as 5HEX (Hexachlorofluorescein) 3IBQ (Iowa Black Quencher). Different other fluorochrome/quencher combinations are possible. Synthesis of TaqMan probes 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. SH-Prot primers (8.3) for the detection of HAV using a TaqMan assay (!72 bp fragment): SH-Prot-A (forward) 20 bases = 5 - ATG GAT GCT GGR GTT CTT AC - 3 SH-Prot-1 (reverse) 22 bases = 5 - ART TGG CAG CAA TTT CTT CAA G -3 SH-Prot probe (8.3) for the detection of HAV using a TaqMan assay: SH-Prot-Q 30 bases = 5 TGA ATG ATG AGA AAT GGA CAG AAA TGA AGG - 3 Standard MixBase Definitions Letter Nucleotides Letter Nucleotides 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 Note 1: Synthesis of oligonucleotide primers can usually be contracted out to a local university or, alternatively, many biotechnology firms offer a custom synthesis service. It is advisable to order oligonucleotides that have been purified by High Pressure/Performance Liquid Chromatography (HPLC) for real-time RT-PCR applications. If assistance is required in this matter, contact the authors. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 17 - Note 2: SH-Prot-Q TaqMan probe was ordered as 5HEX (Hexachlorofluorescein) 3IBQ (Iowa Black Quencher). Different other fluorochrome/quencher combinations are possible. Synthesis of TaqMan probes 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. 10.2 Temperature cycling program for HAV primers and probe systems Using the SH-Poly primers and probe system, the spectrofluorometric thermal cycler program should be set for the following sequence of cycling parameters: Step 1 Reverse transcription 30 minutes 50 C Step 2 Initial PCR activation step 10 minutes 95 C SureStart Taq DNA Polymerase is activated; Reverse Transcriptase is inactivated and the cDNA template is denatured (8.1). Step 3 45 cycles of: Step 3.1 Denaturation 15 seconds 95 C Step 3.2 Annealing + Extension 60 seconds 60 C Note: The use of spectrofluorometric thermal cyclers other than the models stated above or the use of other QRT-PCR reagent kit than the Stratagene Brilliant II QRT-PCR Core Reagent kit 1-Step may alter the performance of the QRT-PCR reactions. It may be necessary, for the user, to optimize the cycling parameters for different models or the concentration of primers, probe or MgCl2. Using the SH-Prot primers and probe system, the spectrofluorometric thermal cycler program should be set for the following sequence of cycling parameters: Step 1 Reverse transcription 30 minutes 50 C Step 2 Initial PCR activation step 10 minutes 95 C SureStart Taq DNA Polymerase is activated; Reverse Transcriptase is inactivated and the cDNA template is denatured (8.1). Step 3 45 cycles of: Step 3.1 Denaturation 15 seconds 95 C Step 3.2 Annealing + Extension 60 seconds 60 C Note: The use of spectrofluorometric thermal cyclers other than the models stated above or the use of other QRT-PCR reagent kit than the Stratagene Brilliant II QRT-PCR Core Reagent kit 1-Step may alter the performance of the QRT-PCR reactions. It may be necessary, for the user, to optimize the cycling parameters for different models or the concentration of primers, probe or MgCl2. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 18 - 10.3 Stratagene Brilliant II QRT-PCR Core Reagent kit, 1-Step (8.1) All stock solutions are stored at -20C until used. The following is a recipe for preparing a large batch equivalent to 102 reactions. Note 1: The protocol for real-time RT-PCR amplification has been established according to the manufacturers instructions provided in the kit manual. Always follow the latest manufacturers instructions provided in Stratagene Brilliant II QRT-PCR Core Reagent kit 1-Step. The most up-to-date manufacturers instructions will prevail over the herein instructions. Note 2: Great care should be taken when working with RNA to avoid contact with ubiquitous RNases. RNases are very stable and are difficult to inactivate. Always store the extracted RNA with 20 units of RNase inhibitor and wear gloves while handling reagents and samples. Use sterile, disposable RNase-free plasticware. Glassware should be treated (cleaned and thoroughly rinsed and baked at 240C for four or more hours before use). Note 3: 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 HAV or HAV RT-PCR products. To avoid any non-specific amplification, the mix should be prepared by putting all the reagents on ice or on a refrigerated rack. Centrifuge before PCR amplification. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 19 - Note: To compensate for non-PCR related variations in fluorescence, some spectrofluorometric thermal cyclers may require the use of ROX as a passive reference dye. If ROX is used as a passive reference dye, dilute 1:500 in nuclease-free PCR-grade water prior to use. RT-PCR Components Initial concentration Stock solutions required for 102 reactions tubes using SH-Poly system Volume per tube Final Concentration RNase-free water - 1313.25 l 12.875 l - 10 X Core RT-PCR buffer 10 X 255.0 l 2.5 l 1 X Magnesium chloride 50 mM 280.5 l 2.75 l 5.5 mM SH-Poly-A primer 10 M 76.5 l 0.75 l 0.3 M SH-Poly-1 primer 10 M 102.0 l 1.0 l 0.4 M SH-Poly-Q probe 10 M 51.0 l 0.5 l 0.2 M dNTP Mix 20 mM (5 mM of each dNTP) 102.0 l 1.0 l 800 M Reverse transcriptase - 102.0 l 1.0 l - Reference dye 1 mM (ROX) Diluted 2 M 38.25 l 0.375 l 0.03 M SureStart Taq DNA Polymerase 5 U/l 25.5 l 0.25 l 0.05 U/l Total Volume 2346.0 l 23.0 l - Distribute per tube 23.0 l - - Template per tube - 2.0 l - Note: If ROX is omitted as a passive reference dye, replace with an equivalent volume of RNase-free water. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 20 - Note: To compensate for non-PCR related variations in fluorescence, some spectrofluorometric thermal cyclers may require the use of ROX as a passive reference dye. If ROX is used as a passive reference dye, dilute 1:500 in nuclease-free PCR-grade water prior to use. RT-PCR Components Initial concentration Stock solutions required for 102 reactions tubes using SH-Prot system Volume per tube Final Concentration RNase-free water - 1287.75 l 12.625 l - 10 X Core RT-PCR buffer 10 X 255.0 l 2.5 l 1 X Magnesium chloride 50 mM 280.5 l 2.75 l 5.5 mM SH-Prot-A primer 10 M 102.0 l 1.0 l 0.4 M SH-Prot-1 primer 10 M 102.0 l 1.0 l 0.4 M SH-Prot-Q probe 10 M 51.0 l 0.5 l 0.2 M dNTP Mix 20 mM (5 mM of each dNTP) 102.0 l 1.0 l 800 M Reverse transcriptase - 102.0 l 1.0 l - Reference dye 1 mM (ROX) Diluted 2 M 38.25 l 0.375 l 0.03 M SureStart Taq DNA Polymerase 5 U/l 25.5 l 0.25 l 0.05 U/l Total Volume 2346.0 l 23.0 l - Distribute per tube 23.0 l - - Template per tube - 2.0 l - Note: If ROX is omitted as a passive reference dye, replace with an equivalent volume of RNase-free water. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 21 - 11. PREPARATION OF THE cDNA CLONE FOR REAL-TIME RT-PCR STANDARD CURVE 11.1 Production of the cDNA fragment using conventional RT-PCR 11.1.1 Amplify HAV RNA with the set of real-time HAV primers (SH-Poly-A and SH-Poly-1 and/or SH-Prot-A and SH-Prot-1) using the OneStep RT-PCR kit from Qiagen with the RT-PCR program described under 9.2. 11.1.2 Add 10.0 l of FCV RNA sample to 40.0 l of conventional RT-PCR reaction mixture. Note: Double the recipe described under 9.3. 11.1.3. After the RT-PCR is completed, analyze the RT-PCR product by agarose gel electrophoresis as described under 7.5. If necessary, the amplicons can be stored at 4C. 11.1.4 Prepare sample for electrophoresis: in a clean microfuge tube, mix 4.0 l of tracking dye (nucleic acid loading buffer 10 X concentrated) and 40 l of RT-PCR product. The amplicons (RT-PCR products) generated by the HAV SH-Poly primers and SH-Prot primers are double stranded DNA fragments of 107 bp and 172 bp, respectively. 11.2 Purification of the RT-PCR product using the QIAquick gel extraction kit (8.7) Note: All reagents used in this section are provided in the kit with the exception of ethanol 96-99%. The protocol for the purification of the RT-PCR product has been established according to the manufacturers instructions provided in the kit manual. Add ethanol (9699%) to Buffer PE before use. Always follow the latest manufacturers instructions provided in the QIAquick gel extraction kit. The most up-to-date manufacturers instructions will prevail over the herein instructions. 11.2.1 Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. 11.2.2 Weigh the gel slice in a colorless microtube. Add 6 volumes of Buffer QG to 1 volume of gel (100 mg 100 l). 11.2.3 Incubate at 50 C for 10 minutes or until the gel slice has completely dissolved. To help dissolve gel, mix by vortexing the tube every 23 min during the incubation. Note: Solubilize agarose completely. For 3% gels, it may be necessary to increase the incubation time. 11.2.4 After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 l of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. Note: The adsorption of DNA to the QIAquick membrane is efficient only at pH 7.5. Buffer QG contains a pH indicator which is yellow at pH 7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding. 11.2.5 Add 1 gel volume of isopropanol to the sample and mix. 11.2.6 Place a QIAquick spin column in a provided 2 ml collection tube. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 22 - 11.2.7 To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 minute at 17900 x g (13000 rpm) in a conventional table-top microcentrifuge at room temperature (23 C +/- 3 C). The maximum volume of the column reservoir is 800 l. For sample volumes of more than 800 l, simply load and spin again. 11.2.8 Discard flow-through and place QIAquick column back in the same collection tube. Collection tubes are reused to reduce plastic waste. 11.2.9 Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 minute at 17900 x g (13000 rpm). 11.2.10 To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 minute at 17900 x g (13000 rpm). 11.2.11 Discard the flow-through and centrifuge the QIAquick column for an additional 1 minute at 17900 x g (13000 rpm). Note: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. 11.2.12 Place QIAquick column into a clean 1.5 ml microcentrifuge tube. 11.2.13 To elute DNA, add 50 l of Buffer EB (10 mM TrisCl, pH 8.5) or water (pH 7.08.5) to the center of the QIAquick membrane, let stand for 1 minute and centrifuge the column for 1 minute at 17900 x g (13000 rpm). Note: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 l from 50 l elution buffer volume. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at 20 C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM TrisCl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions. 11.2.14 Verify the purified DNA by agarose gel electrophoresis as described under 7.5 by loading 9 l of purified product and 1 l of tracking dye (nucleic acid loading buffer 10 X concentrated). 11.3 Cloning reaction with TOPO TA Cloning kit and transformation of competent cells (8.9) Note 1: All reagents used in this section are provided in the kit with the exception of LB medium and LB agar plates containing the appropriate antibiotic and X-Gal. The protocol for the transformation of E. coli with TOPO TA Cloning kit has been established according to the manufacturers instructions provided in the kit manual. Always follow the latest manufacturers instructions provided in the TOPO TA Cloning user Manual. The most up-to-date manufacturers instructions will prevail over the herein instructions. Note 2: Transformation of E. coli can be performed in two ways, chemically or by electroporation. Refer to Invitrogen user manual for the chemical transformation. 11.3.1 Just before beginning, warm the vial of S.O.C medium to room temperature (23 C +/- 3 C). OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 23 - 11.3.2 Warm selective plates (LB plate containing 50 g/ml of kanamycin) at 37 C for 30 minutes. 11.3.3 Spread 40 l of 40 mg/ml X-Gal in dimethylformamide (DMF) on each LB agar plate and incubate at 37 C until ready for use. 11.3.4 Place the electroporation cuvettes on ice. 11.3.5 Thaw on ice 1 vial of One Shot TOP10 Electrocompetent E. coli cells. 11.3.6 Dilute the salt solution 4 fold by adding 15 l of water to 5 l of salt solution. 11.3.7 Set up the TOPO cloning reaction by adding 4 l of purified PCR product to 1 l of diluted salt solution and 1 l of PCR2.1-TOPO vector. 11.3.8 Mix gently and incubate for 5 minutes at room temperature. 11.3.9 Place the reaction on ice. 11.3.10 Add 2 l of the TOPO cloning reaction into a vial of One Shot TOP10 Electrocompetent E. coli cells and mix gently. Note: Do not mix by pipetting up and down. 11.3.11 Carefully transfer solution to a 0.1 cm cuvette to avoid formation of bubbles. 11.3.12 Electroporate your sample using your own protocol and electroporator. 11.3.13 Immediately add 250 l of room temperature S.O.C medium. 11.3.14 Transfer the solution to a 15 ml snap cap tube (e.g., Falcon) and shake for at least 1 hour at 37 C to allow expression of the antibiotic resistance genes. 11.3.15 Spread 10-50 l from each transformation on a pre-warmed LB plate containing X-Gal and 50 g/ml kanamycin and incubate overnight at 37 C. 11.3.16 Take 10 white colonies and culture them overnight at 37 C in 5 ml of LB medium containing 50 g/ml kanamycin. 11.3.17 Isolate plasmid DNA to confirm the presence of the corresponding HAV insert. 11.4 Isolation of plasmid DNA from E. coli using NucleoSpin Plasmid kits (8.4) Note: All reagents used in this section are provided in the kit with the exception of with the exception of ethanol 96-99% and 1.5 ml microcentrifuge tubes. The protocol for the isolation of plasmid DNA from E. coli has been established according to the manufacturers instructions provided in the kit manual. Always follow the latest manufacturers instructions provided in the NucleoSpin Plasmid kits user Manual. The most up-to-date manufacturers instructions will prevail over the herein instructions. OPFLP-07 March 2010 Published on the Food Directorates (Health Canada) website at http:/www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index-eng.php - 24 - SAFETY NOTE: BUFFERS A3 AND AW CONTAIN GUANIDINE HYDROCHLORIDE WHICH IS HIGHLY TOXIC. TAKE APPROPRIATE SAFETY MEASURES AND WEAR GLOVES WHEN HANDLING. DISPOSE OF ALL SOLUTIONS, BUFFERS AND REAGENTS ACCORDING TO THE WASTE-DISPOSAL GUIDELINES. 11.4.1 Before beginning, add the entire contents of the provided vial of RNase A to the bottle of Buffer A1. Mix well and store at 4 C for up to 6 months. 11.4.2 Add 96-99% non-denatured ethanol to Buffer A4 before use. The appropriate volume of ethanol is printed on the Buffer A4 bottle. 11.4.3 Pipet 1.5 ml of saturated E. coli culture and centrifuge at 11000 x g for 30 seconds

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