Detection of feline caliciviruses using the conventional and real-time reverse-transcriptase polymerase chain reaction

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Laboratory Procedure OPFLP-06
March 2010

Health Products and Food Branch

Ottawa

Detection of feline caliciviruses using the conventional and real-time reverse-transcriptase polymerase chain reaction

Kirsten Mattison¹, Michelle Plante¹, Sabah Bidawid¹, Cesar bin Kingombe¹, Alain Houde², Pierre Ward²

Microbiological Methods Committee
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204E
HPFB, Ottawa, Ontario, K1A 0K9

e-mail: Micro_methods_committee@hc-sc.gc.ca

1. Application

This method is applicable to the amplification and detection of feline calicivirus (FCV) RNA by conventional and real-time (TaqMan®) reverse-transcriptase polymerase chain reaction (RT-PCR) following the extraction of nucleic acids from a sample. This method will not differentiate between infectious and non-infectious viruses.

2. Description

This protocol is routinely used to amplify and detect FCV RNA. FCV may be used as a positive processing control (internal control for extraction and amplification) for all RNA viruses in various studies involving food and waterborne viruses. In such instances, FCV should be used in a final concentration of ≤1000 PFU/gram of food matrix.

3. Principle

FCV, as a positive processing control, can be artificially inoculated in food, clinical or environmental samples. Total RNA is extracted from the food, clinical or environmental sample using an appropriate procedure or a cell free sample (for instance, FCV 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 on the primers used. FCV amplified fragments are confirmed by HpaII restriction endonuclease digestion after gel electrophoresis visualization for conventional RT-PCR or by fluorescence reading of the TaqMan® probe degradation by the Taq polymerase exonuclease activity during the amplification process for real-time RT-PCR.

4. Definition of terms

4.1 See Appendix A of Volume 3.

5. Virus Stocks

5.1 FCV is propagated in CrFK cells, aliquoted in 2 ml cryogenic vials and stored at -70°C (See OPFLP-08).

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® Mastercyler® 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 to 10 µI, 10 to 100 µI, and 100 to 1000 µI with specific filtered pipet tips.

6.8 Microcentrifuge for microfuge tubes -1.5 ml/2.0 ml capacity and a speed range of 1000 to 14000 rpm (20000 x g max) (Labnet International Spectrafuge 16 M or equivalent).

6.9 Standard heatblocks (VWR scientific products or equivalent) or waterbath capable of accommodating 1.5 ml microfuge tubes and capable of maintaining a temperature of 37-50°C.

Note: 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 water bath may be at 37°C +/- 1°C. For all other temperatures it may be +/- 2°C.

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 -20°C and -70°C.

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, OneStep (Stratagene® product number 600810 or equivalent).

6.19 Non-denatured ethanol 96- 99% (room temperature 23°C +/- 3°C).

6.20 FCV primers (CBK-1 and CBK-2) for the detection of FCV by conventional RT-PCR (See section 9.1 for DNA sequences).

6.21 HpaII restriction endonuclease and buffer (Invitrogen™ product number 15209-018, or equivalent).

6.22 EcoRI restriction endonuclease and buffer (Invitrogen™ product number 15202-021, or equivalent).

6.23 BamHI restriction endonuclease and buffer (Invitrogen™ product number 15201-031, or equivalent).

6.24 FCV primers (SH-FCV3-Q-A and SH-FCV3-Q-1) and TaqMan® probe (SH-FCV3-P) for the detection of FCV by real-time RT-PCR (See section 10.1 for DNA sequences).

6.25 QIAquick® gel extraction kit (Qiagen® product number 28704 or 28706, or equivalent).

6.26 TOPO® TA Cloning kit (with PCR®2.1-TOPO® vector) with One Shot® TOP10 Electrocomp™ E. coli (Invitrogen™ product number K4560-01 or K4560-40, or equivalent).

6.27 X-GAL (5-Bromo-4-Chloro-3-Indolyl-ß-D-Galactopyranoside) (BioShop product number XGA001 or equivalent).

6.28 Electroporation system (Bio-Rad Gene Pulser™ product or equivalent).

6.29 Electroporation cuvettes Gene Pulser/MicroPulser cuvettes, 0.1 cm gap, (Bio-Rad product number 165-2089 or equivalent).

6.30 Kanamycin, 10 mg/ml (Invitrogen™ product number 18161-054 or equivalent).

6.31 LB broth Miller (BioShop product number LBL407-50 or equivalent).

6.32 NucleoSpin® Plasmid kit (Macherey Nagel product number MCN540788050 or MCN540788250, or equivalent).

6.33 Spectrophotometer (Thermo Scientific NanoDrop™ 1000 or equivalent).

6.34 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) (BlueJuice™ Gel Loading Buffer, Invitrogen™ product number 10816-015 or equivalent).

RNase inhibitor (RNaseOUT™ Recombinant Ribonuclease Inhibitor 40 U/µI, 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 Handling of sample units

7.1.1 FCV stocks should remain frozen at -70°C until needed.

7.1.2 During storage and transport keep the RNA extracted from sample units frozen (-70°C).

7.1.3 Extracted RNA should be kept on ice while preparing the conventional or the real-time RT-PCR reactions.

7.2 Preparation for analysis

7.2.1 Ensure that all reagents needed are available.

7.2.2 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 FCV 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.

7.2.3 FCV titre could be estimated using conventional RT-PCR. The titre would then be expressed in RT-PCR units. Thaw a 2 ml cryogenic vial containing FCV. Once thawed, make 10-fold dilution series of the virus by adding 100 µl of virus stock to 900 µl of RNase-free water in a 1.5 ml microfuge tube (this is the 10^-1 dilution). Repeat this step by taking 100 µl of the 10^-1 viral dilution and add to 900 µl 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.

7.2.4 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 SH-FCV3-Q-A and SH-FCV3-Q-1 primer set in a 5 ng/ml salmon sperm DNA solution. Once thawed, make 10-fold dilution series of the cDNA plasmid by adding 10 µI of plasmid stock to 90 µI 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 µI of the 10^-1 cDNA plasmid dilution and add to 90 µI of RNase-free water containing salmon 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.

7.3 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 manufacturer’s instructions provided in the kit manual. AVL buffer must be completely dissolved prior to being used. Always follow the latest manufacturer’s instructions provided in the QIAamp® Viral RNA Mini kit. The most up-to-date manufacturer’s 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 > 240°C for four hours or more before use).

7.3.1 Pipet 560 µI 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.

7.3.2 Add 140 µI 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 WASTEDISPOSAL GUIDELINES.

7.3.3 Pulse-vortex microfuge tubes for 15 seconds.

7.3.4 Incubate the samples for 10 minutes at room temperature (23°C +/- 3°C).

7.3.5 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).

7.3.6 Add 560 µI of non-denatured ethanol (96 - 99%) to the samples.

7.3.7 Pulse-vortex microfuge tubes for 15 seconds.

7.3.8 Transfer 630 µI of the solution to a QIAamp® Mini spin column (in a 2 ml collection tube) without wetting the rim.

7.3.9 Close the caps and centrifuge the spin columns at 6000 x g for 1 minute.

7.3.10 Transfer each spin column to a clean 2-ml collection tube.

7.3.11 Discard the previously used collection tubes.

7.3.12 Transfer the remaining 630 µI of each solution to its respective spin column.

7.3.13 Centrifuge at 6000 x g for 1 minute.

7.3.14 Repeat steps 7.3.10 and 7.3.11.

7.3.15 Add 500 µI of buffer AW1 to each spin column and close the lids.

7.3.16 Centrifuge at 6000 x g for 1 minute.

7.3.17 Transfer each spin column to a clean 2 ml collection tube.

7.3.18 Discard previously used collection tube.

7.3.19 Add 500 µI 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.

7.3.20 Centrifuge at 20000 x g for 3 minutes.

7.3.21 Place each spin column in a 1.5 ml microfuge tube.

7.3.22 Discard previously used collection tubes.

7.3.23 Add 60 µI of buffer AVE to each spin column.

Note: Alternatively, the addition of 2 X 30 µI 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 1min) (8.3).

7.3.24 Close the caps and incubate at room temperature (23°C +/- 3°C) for 1 minute.

7.3.25 Centrifuge at 6000 x g for 1 minute.

7.3.26 Discard spin columns and close lids of 1.5 ml microfuge tubes.

7.3.27 Add 20 units of RNase inhibitor (0.5 µI of RNaseOUT™ 40 U/µI) to the extracted RNA.

7.3.28 Use RNA directly in RT-PCR, real-time RT-PCR or store at - 70°C until needed.

7.4 Conventional RT-PCR method for the amplification of FCV RNA

7.4.1 Amplify RNA from each extracted RNA sample with the set of FCV primers (CBK-1 and CBK-2) using the OneStep RT-PCR kit from Qiagen®.

7.4.2 Add 1.0 µI of each extracted RNA sample to 24.0 µI of RT-PCR reaction mixture (9.3).

7.4.3. Set up a negative control by adding 1.0 µI of RNase-free water to 24.0 µI of the same RTPCR reaction mixture (9.3). For positive controls, add 1.0 µI of FCV RNA to 24.0 µI 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 4°C until analysis and/or confirmed by digestion with HpaII restriction endonuclease (7.7).

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 45°C then add the concentrated ethidium bromide (EtBr) solution to obtain a final concentration of 0.5 µg/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 (8.6).

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 WASTEDISPOSAL GUIDELINES.

7.5.3. Pour into a gel tray. Avoid bubble formation or bubble trapping. Add a well-forming comb and allow the gel to solidify for about 20 to 30 minutes.

7.5.4. Prepare samples for electrophoresis: in clean microfuge tubes, mix 1.2 µI of tracking dye (nucleic acid loading buffer 10 X concentrated) into each microfuge tubes containing 10 µI 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 µI) (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 µg/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 FCV CBK-1 and CBK-2 primers are double stranded DNA fragments of 218 bp. 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 / 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-analyzed.

7.6.4 Any band corresponding to the positive control occurring in the negative control with the FCV 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-analyzed using a new reagent batch.

7.6.5 Any test sample showing a distinct band with the FCV primers, corresponding to its positive control, is considered as a presumptive positive. The PCR product should be confirmed by digestion with HpaII restriction endonuclease (7.7).

7.7 Restriction endonuclease digestion

7.7.1 Add 10 µI of each amplicon (7.4) and 10 µI of RNase-free water to separate 1.5 ml microfuge tubes.

7.7.2 Add 2.4 µI of restriction endonuclease buffer (provided with restriction endonuclease) and 2.0 µI of HpaII (10 U/µI) restriction endonuclease to each microfuge tube.

7.7.3 Incubate overnight in a waterbath or heatblock at 37°C.

7.7.4 After the reaction has been completed, analyze the digested PCR product by agarose gel electrophoresis (7.5).

7.7.5 The two DNA fragments generated by the HpaII restriction endonuclease are 114 and 104 bp in molecular size. Therefore, bands that have a molecular size of 114 and 104 bp as compared to a DNA molecular size marker (e.g., 100 bp DNA ladder) run on the same gel can be confirmed as being FCV amplicons.

Note: The two DNA fragments of 114 and 104 bp generated by the digestion with HpaII restriction endonuclease may appear as co-migrating on a 2.0% (w/v) agarose gel.

7.8 Calculating RT-PCR units

7.8.1 After reading the RT-PCR results (7.6), determine the highest FCV dilution (7.2.3) showing a positive result (7.6.5) which is called the end-point dilution.

7.8.2 Once determined, take the reciprocal of this dilution to give RT-PCR units per quantity used for the RT-PCR (i.e., 1.0 µI).

7.8.3 To obtain a concentration per ml, multiply the concentration obtained in step 7.8.2 by a factor that will give you 1000 µI or 1 ml (i.e., if 1.0 µI of RNA was used for the RT-PCR and 10^-4 was the highest dilution in which a band was observed, multiply 10⁴ by 1000). Therefore, the RT-PCR units are 1.0 x 10⁷ RT-PCR units per ml.

Note: Viral RNA titres are usually expressed as RT-PCR units per ml.

7.9 Real-time RT-PCR method for the amplification of FCV RNA

7.9.1 Amplify RNA from each extracted RNA sample in duplicate with the set of FCV primers and TaqMan® probe (SH-FCV3-Q-A, SH-FCV3-Q-1 and SH-FCV3-P) 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.

7.9.2 Add 2.0 µI of each extracted RNA sample or clone used for the standard curve to 23.0 µI of real-time RT-PCR reaction mixture (10.3).

7.9.3. Set up reaction controls in triplicate. For the No Template Control (NTC), add 2.0 µI of RNase-free water to 23.0 µI of the same real-time RT-PCR reaction mixture (10.3). As positive control, add 2.0 µI of FCV RNA that has been previously confirmed as positive in other experiments or cDNA from the corresponding clone to 23.0 µI 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 FCV genomic RNA.

7.9.4 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 (60°C).

7.9.5 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 4°C for further analysis.

7.10 Reading real-time RT-PCR results

7.10.1 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 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.

7.10.2 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).

7.10.3 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.

7.10.4 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 reanalyzed.

7.10.5 Any Ct occurring in the negative control with the FCV 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-analyzed using a new reagent batch.

7.10.6 Any sample showing a Ct with the FCV 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.

7.11 Calculating genomic equivalent units

7.11.1 Unknown sample Ct value must fall within 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 the standard curve.

7.11.2 To obtain the initial concentration in genomic equivalents per µI, divide the concentration obtained in step 7.11.1 by the volume of RNA used as template (i.e., if 2.0 µI 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 µI). Therefore, the concentration is 2.0 x 10⁶ genomic equivalents per µI).

Note: Viral RNA concentrations are usually expressed as genomic equivalent per µI 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: http://www.stratagene.com/manuals/600810.pdf

8.2 NucleoSpin® Plasmid kits user Manual. March 2008. Available at: http://www.mnnet. com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purificatio n/UM_pDNA_NS.pdf

8.3 QIAamp® Viral RNA Mini Handbook. December 2007. Available at: http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000199

8.4 Qiagen® OneStep RT-PCR Kit Handbook. February 2008. Available at: http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000223

8.5 QIAquick® Spin Handbook. March 2008. pp. 25-26. Available at: http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000252

8.6 SYBR™ Safe DNA gel stain. July 2006. Available at: http://probes.invitrogen.com/media/pis/mp33100.pdf

8.7 TOPO TA Cloning® user Manual. April 2006. Available at: http://tools.invitrogen.com/content/sfs/manuals/topota_man.pdf

9. Conventional RT-PCR Reagents and Temperature Cycling Program

9.1 RT-PCR primers

FCV primers for the detection of FCV (218 bp fragment):
CBK-1 (forward) 20 bases = 5' - GGA GGC GCG ATC TTC AGT AT - 3'
CBK-2 (reverse) 20 bases = 5' - GCA TAA CTC GTC GGA GGT GT -3'

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 FCV primers

Using the FCV 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 (8.4).
Step 3 32 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).

9.3 Qiagen® OneStep RT-PCR kit (8.4)

All stock solutions are stored at -20°C 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 manufacturer’s instructions provided in the kit manual. Always follow the latest manufacturer’s instructions provided in Qiagen® OneStep RT-PCR kit. The most up-to-date manufacturer’s 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 > 240°C 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 FCV or FCV 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 µI of RTPCR mix in 200 µI tubes are used.

RT-PCR Components	Initial concentration	Stock solutions required for 20 reactions tubes using CBK primers	Volume per tube	Final Concentration
RNase-free water	---------	280.0 µI	14.0 µI	--------
5 X QIAGEN® OneStep RT-PCR buffer	5 X	100.0 µI	5.0 µI	1 X
dNTP Mix	10 mM of each dNTP	20.0 µI	1.0 µI	400 µM
CBK-1 primer	10 µM	30.0 µI	1.5 µI	0.6 µM
CBK-2 primer	10 µM	30.0 µI	1.5 µI	0.6 µM
QIAGEN® OneStep RT-PCR Enzyme Mix	---------	20.0 µI	1.0 µI	--------
Total Volume		480.0 µI	24.0 µI	--------
Distribute per tube		24.0 µI	--------	--------
Template per tube		--------	1.0 µI	--------

10. Real-Time RT-PCR Reagents and Temperature Cycling Program

10.1 Real-time RT-PCR primers and probe

SH-FCV primers for the detection of FCV using a TaqMan® assay (85 bp fragment):
SH-FCV3-Q-A (forward) 21 bases = 5' – GAC ACC TCC GAC GAG TTA TGC - 3'
SH-FCV3-Q-1 (reverse) 18 bases = 5' – CCG GGT GGG ACT GAG TGG -3'

SH-FCV probe for the detection of FCV using a TaqMan® assay:
SH-FCV3-P 30 bases = 5' – CGC CTT ACG GAT ATG AGC AGC CAC ATT AAC - 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-FCV3-P TaqMan® probe was ordered as 5’HEX™ (Hexachlorofluorescein)– 3’IBQ™ (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 FCV primers and probe system

Using the SH-FCV3 primer 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- OneStep 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.

10.3 Stratagene® Brilliant® II QRT-PCR Core Reagent kit, OneStep (8.1)

All stock solutions are stored at -20°C 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 manufacturer’s instructions provided in the kit manual. Always follow the latest manufacturer’s instructions provided in Stratagene® Brilliant® II QRT-PCR Core Reagent kit, 1-Step. The most up-to-date manufacturer’s 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 > 240°C 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 FCV or FCV 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.

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-FCV3 system	Volume per tube	Final Concentration
RNase-free water	---------	1364.25 µI	13.375 µI	--------
10 X Core RT-PCR buffer	10 X	255.0 µI	2.5 µI	1 X
Magnesium chloride	50 mM	255.0 µI	2.5 µI	5.0 mM
SH-FCV3-Q-A primer	10 µM	76.5 µI	0.75 µI	0.3 µM
SH-FCV3-Q-1 primer	10 µM	76.5 µI	0.75 µI	0.3 µM
SH-FCV3-P probe	10 µM	51.0 µI	0.5 µI	0.2 µM
dNTP Mix	20 mM (5 mM of each dNTP)	102.0 µI	1.0 µI	800 µM
Reverse transcriptase	---------	102.0µI	1.0 µI	--------
Reference dye 1 mM (ROX™)	Diluted 2 µM	38.25 µI	0.375 µI	0.03 µM
SureStart® Taq DNA Polymerase	5 U/µI	25.5 µI	0.25 µI	0.05 U/µI
Total Volume		2346.0 µI	23.0 µI	--------
Distribute per tube		23.0 µI	--------	--------
Template per tube		--------	2.0 µI	--------

Note: If ROX™ is omitted as a passive reference dye, replace with an equivalent volume of RNasefree water.

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 FCV RNA with the set of real-time FCV primers (SH-FCV3-Q-A and SH-FCV3-Q- 1) using the OneStep RT-PCR kit from Qiagen® with the RT-PCR program described in 9.2.

11.1.2 Add 2.0 µI of FCV RNA sample to 48.0 µI of conventional RT-PCR reaction mixture.

Note: Double the recipe described in 9.3.

11.1.3. After the RT-PCR is completed, analyze the RT-PCR product by agarose gel electrophoresis as described in 7.5 but on a 3.0% (w/v) agarose gel. If necessary, the amplicons can be stored at 4°C.

11.1.4 Prepare sample for electrophoresis: in a clean microfuge tube, mix 4.0 µI of tracking dye (nucleic acid loading buffer 10 X concentrated) and 40 µI of RT-PCR product. The amplicon (RT-PCR products) generated by the FCV SH-FCV3-Q-A and SH-FCV3-Q-1 primers is a double stranded DNA fragment of 85 bp.

11.2 Purification of the RT-PCR product using the QIAquick® gel extraction kit (8.5)

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 manufacturer’s instructions provided in the kit manual. Add ethanol (96–99%) to Buffer PE before use. Always follow the latest manufacturer’s instructions provided in the QIAquick® gel extraction kit. The most up-to-date manufacturer’s 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 µI).

11.2.3 Incubate at 50°C for 10 min or until the gel slice has completely dissolved. To help dissolve gel, mix by vortexing the tube every 2–3 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 µI 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.

11.2.7 To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature (23°C +/- 3°C). The maximum volume of the column reservoir is 800 µI. For sample volumes of more than 800 µI, 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 min at 17,900 x g (13,000 rpm).

11.2.10 To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min at 17,900 x g (13,000 rpm).

11.2.11 Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 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 µI of Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane, let stand for 1 min and centrifuge the column for 1 min at 17,900 x g (13,000 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 µI from 50 µI 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 Tris·Cl, 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 in 7.5 by loading 9 µI of purified product and 1 µI of tracking dye (nucleic acid loading buffer 10 X concentrated) on a 3.0 % (w/v) agarose gel.

11.3 Cloning reaction with TOPO® TA Cloning kit and transformation of competent cells (8.7)

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 manufacturer’s instructions provided in the kit manual. Always follow the latest manufacturer’s instructions provided in the TOPO® TA Cloning user Manual. The most up-to-date manufacturer’s 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).

11.3.2 Warm selective plates (LB plate containing 50 µIg/ml of kanamycin) at 37°C for 30 min.

11.3.3 Spread 40 µI 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 µI of water to 5 µI of salt solution.

11.3.7 Set up the TOPO® cloning reaction by adding 4 µI of purified PCR product to 1 µI of diluted salt solution and 1 µI of PCR®2.1-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 µI 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 µI 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 µI from each transformation on a pre-warmed LB plate containing X-Gal and 50 µg/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 µg/ml kanamycin.

11.3.17 Isolate plasmid DNA to confirm the presence of the FCV insert.

11.4 Isolation of plasmid DNA from E. coli using NucleoSpin® Plasmid kits (8.2)

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 manufacturer’s instructions provided in the kit manual. Always follow the latest manufacturer’s instructions provided in the NucleoSpin® Plasmid kits user Manual. The most up-to-date manufacturer’s instructions will prevail over the herein instructions.

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 11,000 x g for 30 s to pellet bacterial cells.

11.4.4 Discard the supernatant.

11.4.5 Resuspend the pellet in 250 µI of Buffer A1 by vigorous vortexing.

Note: Check buffer A2 for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30 - 40°C until precipitate is dissolved completely. Cool buffer down to room temperature (23°C +/- 3°C).

11.4.6 Add 250 µI of Buffer A2 to the suspension. Mix gently by inverting the tube 6-8 times, and incubate at room temperature for 5 min.

Note: Do not vortex to avoid the release of contaminating chromosomal DNA into the suspension.

11.4.7 Add 300 µI of Buffer A3 to the suspension. Mix gently by inverting the tube 6-8 times.

11.4.8 Centrifuge the suspension at 11,000 x g in a microcentrifuge for 5 min. Repeat this step if the supernatant is not clear.

11.4.9 Place a NucleoSpin Plus column in a 2 ml collection tube and load the supernatant from 11.4.8 into the spin column.

11.4.10 Centrifuge at 11,000 x g for 1 min and discard the flowthrough.

11.4.11 Wash the NucleoSpin column with 500 µI of Buffer AW.

11.4.12 Centrifuge at 11,000 x g for 1 min and discard the flowthrough.

11.4.13 Reinsert the NucleoSpin column in the 2-ml collection tube and add 600 µI of Buffer A4 (with ethanol) to the NucleoSpin column.

11.4.14 Centrifuge at 11,000 x g for 1 min and discard the flowthrough.

11.4.15 Recentrifuge at 11,000 x g for 2 min to remove residual ethanol from the silica membrane inside the spin column.

11.4.16 Place the NucleoSpin column in a 1.5-ml or 2-ml microcentrifuge tube (not included with kit). Elute the DNA by adding 50 µI of Buffer AE (5 mM Tris-HCl, pH 8.5) to the column. Incubate 1 minute at room temperature (23°C +/- 3°C).

11.4.17 Centrifuge at 11,000 x g for 1 min. The plasmid DNA is now collected in Buffer AE at the bottom of the centrifuge tube.

11.5 Confirmation of the presence of the FCV insert

11.5.1 Add 2.0 µI of purified DNA plasmid (11.4.17), 2.0 µI of restriction endonuclease buffer (provided with restriction endonuclease), 15.0 µI of RNase/DNase-free water and 1.0 µI of EcoRI (10 U/µI) restriction endonuclease in a microfuge tube.

11.5.2 Incubate overnight in a waterbath or heatblock at 37°C.

11.5.3 After the reaction has been completed, prepare sample for electrophoresis. In a clean microfuge tube, mix 10 µI of the digested plasmid product and 1 µI of tracking dye (nucleic acid loading buffer 10 X concentrated). Analyze by agarose gel electrophoresis (7.5) on a 3.0 % (w/v) agarose gel.

11.5.4 The two DNA fragments generated by the EcoRI restriction endonuclease are 3921 bp (plasmid) and 85 bp (FCV cDNA) in molecular size. Therefore, bands that have a molecular size of 85 bp as compared to a DNA molecular size marker (e.g., 100 bp DNA ladder) run on the same gel can be confirmed as being FCV cDNA.

Note: However, it is advisable to sequence your insert to confirm its integrity. Your construct may be sequenced using M13 forward and reverse primers. DNA sequencing can usually be contracted out to a local university or, alternatively, many biotechnology firms offer a custom sequencing service. If assistance is required in this matter, contact the authors.

11.6 Plasmid linearization for being used as a standard

11.6.1 Add 35.0 µI of purified DNA plasmid (11.4.17), 12.0 µI of restriction endonuclease buffer (provided with restriction endonuclease), 65.0 µI of RNase/DNase-free water and 10.0 µI of BamHI (10 U/µI) restriction endonuclease in a microfuge tube.

11.6.2 Incubate 4 hours in a waterbath or heatblock at 37°C.

11.6.3 Inactivate the restriction endonuclease by incubating 20 min in a waterbath or heatblock at 55°C.

11.6.4 After the reaction has been completed, prepare sample for electrophoresis. In a clean microfuge tube, mix 2.0 µI of the digested plasmid product, 8.0 µI of RNase/DNase-free water and 1 µI of tracking dye (nucleic acid loading buffer 10 X concentrated). Analyze by agarose gel electrophoresis (7.5) on a 2.0 % (w/v) agarose gel.

11.6.5 The DNA fragment generated by the BamHI restriction endonuclease is 4016 bp (3931 bp plasmid + 85 bp FCV cDNA) in molecular size.

Note: Only one band should appear on your agarose gel to confirm the linearization of your cDNA clone.

11.7 Standard curve preparation for real-time RT-PCR

11.7.1 Determine the concentration in g/µI of your linear DNA plasmid stock (11.6.3) with a spectrophotometer.

11.7.2 Evaluate the molar mass:
bp length (plasmid + FCV insert) x 649 g/mol [molar mass for 1 kb]
(3931 bp + 85 bp) x 649 g/mol = 2.606 X 10⁶ g/mol

11.7.3 Evaluate the copy number (genomic equivalents)/µI:
copy number/µI = [(concentration of linearized plasmid g/µI) / (molar mass)] x (6.023 x 10²³)

example: If the concentration of linearized plasmid is 143.89 ng/µI then 1.439 X 10^-7 g/µI

copy number/µI = [(1.439 X 10^-7 g/µI) / (2.606 X 10⁶ g/mol)] x (6.023 x 10²³)
copy number/µI = 33.259 x 10⁹ or 3.326 x 10¹⁰

11.7.4 Dilute your linear plasmid stock in RNase/DNase-free water to obtain a concentration of 1 x 10⁹ copies (or genomic quivalents)/µI.

11.7.5 Generate standard curves as described in 7.2.4.

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, CanadaJ2S 8E3