SOP: Detection of Feline Caliciviruses by Conventional and Real-Time Reverse-Transcriptase Polymerase Chain Reaction

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Feline calicivirus (FCV) RNA is detected after extraction from a food, clinical or environmental sample, or from a cell-free FCV stock suspension, using the QIAamp viral RNA Mini Kit. The extracted RNA is amplified by conventional reverse-transcriptase polymerase chain reaction (RT-PCR) with primers CBK-1 and CBK-2, and/or by real-time (TaqMan) RT-PCR with primers SH-FCV3-Q-A and SH-FCV3-Q-1 and probe SH-FCV3-P. Conventional RT-PCR products are confirmed by HpaII restriction endonuclease digestion after agarose gel electrophoresis; real-time RT-PCR products are confirmed by fluorescence from degradation of the TaqMan probe during amplification. FCV may also serve as a positive processing control for all RNA viruses in food and waterborne-virus studies, at a final concentration of at least 1000 PFU per gram of food matrix. The method does not differentiate between infectious and non-infectious viruses.

2. Apparatus required

• Thermal cycler (Eppendorf Mastercycler Gradient or equivalent)
• Spectrofluorometric thermal cycler (Stratagene Mx series or equivalent)
• Microwave oven or hot plate
• Submarine gel casting tray and buffer reservoir, power pack, and appropriate comb
• Shortwave UV light table (transilluminator) for visualizing stained DNA in agarose gels
• Photo documentation system (optional), 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
• Adjustable micropipettors covering 0.5 to 10 µL, 10 to 100 µL, and 100 to 1000 µL, with specific filtered pipette tips
• Microcentrifuge for 1.5 mL / 2.0 mL microfuge tubes, 1000 to 14000 rpm (20000 × g maximum) (Labnet International Spectrafuge 16 M or equivalent)
• Standard heat blocks (VWR scientific products or equivalent) or water bath accommodating 1.5 mL microfuge tubes, capable of maintaining 37 to 50°C
• Vortex mixer
• Sterile microfuge tubes, 1.5 mL and 2.0 mL capacity, and/or 96-well plate
• PCR tubes, thin-wall 0.2 mL or 0.5 mL, strip or 96-well plate (depending on the thermal cycler model)
• Timer
• Container for ice or cooling rack
• Freezers capable of maintaining −20°C and −70°C
• Electroporation system (Bio-Rad Gene Pulser product or equivalent)
• Electroporation cuvettes, Gene Pulser/MicroPulser, 0.1 cm gap (Bio-Rad product number 165-2089 or equivalent)
• Spectrophotometer (Thermo Scientific NanoDrop 1000 or equivalent)

3. Reagents and kits

• QIAamp viral RNA extraction kit (Qiagen product number 52904 or 52906, or equivalent)
• Qiagen OneStep RT-PCR kit (Qiagen product number 210210 or 210212, or equivalent)
• Stratagene Brilliant II QRT-PCR Core Reagent kit, OneStep (Stratagene product number 600810 or equivalent)
• Non-denatured ethanol, 96 to 99% (room temperature, 23°C ± 3°C)
• FCV primers CBK-1 and CBK-2 for conventional RT-PCR (see section 6)
• HpaII restriction endonuclease and buffer (Invitrogen product number 15209-018, or equivalent)
• EcoRI restriction endonuclease and buffer (Invitrogen product number 15202-021, or equivalent)
• BamHI restriction endonuclease and buffer (Invitrogen product number 15201-031, or equivalent)
• FCV primers SH-FCV3-Q-A and SH-FCV3-Q-1 and TaqMan probe SH-FCV3-P for real-time RT-PCR (see section 6)
• QIAquick gel extraction kit (Qiagen product number 28704 or 28706, or equivalent)
• TOPO TA Cloning kit (with pCR2.1-TOPO vector) with One Shot TOP10 Electrocompetent E. coli (Invitrogen product number K4560-01 or K4560-40, or equivalent)
• X-GAL (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside) (BioShop product number XGA001 or equivalent)
• Kanamycin, 10 mg/mL (Invitrogen product number 18161-054 or equivalent)
• LB broth Miller (BioShop product number LBL407-50 or equivalent)
• NucleoSpin Plasmid kit (Macherey Nagel product number MCN540788050 or MCN540788250, or equivalent)
• 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, 10× (BlueJuice Gel Loading Buffer, Invitrogen product number 10816-015 or equivalent)
• RNase inhibitor (RNaseOUT Recombinant Ribonuclease Inhibitor, 40 U/µL, Invitrogen product number 10777-019 or equivalent)
• 0.5× TBE (Tris-Borate-EDTA) or 1× 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)

Reagent preparations

• Salmon Sperm DNA Solution, 10 mg/mL
• Use as supplied

4. Procedure

Sample handling and virus stocks

Keep FCV stocks frozen at −70°C until needed (propagate FCV in CrFK cells and aliquot into 2 mL cryogenic vials)

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During storage and transport, keep the RNA extracted from sample units frozen (−70°C)

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Keep extracted RNA on ice while preparing the conventional or real-time RT-PCR reactions

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Ensure that all reagents needed are available

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Prepare and verify the suitability of all controls — for both conventional and real-time RT-PCR, include a negative amplification control (water as template) and a positive amplification control (previously confirmed FCV RNA or the corresponding cDNA clone) specific to the primers used

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If estimating the FCV titre by conventional RT-PCR, make a 10-fold dilution series of a thawed 2 mL FCV vial — add 100 µL of virus stock to 900 µL of RNase-free water in a 1.5 mL microfuge tube (the 10⁻¹ dilution), then transfer 100 µL of that dilution to 900 µL of RNase-free water (the 10⁻² dilution), and repeat until the 10⁻⁸ dilution is reached; the titre is then expressed in RT-PCR units

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If generating a standard curve for real-time RT-PCR, make 10-fold serial dilutions (10⁸ to 10⁰ genomic equivalents) in triplicate of the purified linear cDNA plasmid (prepared as in section 5) — add 10 µL of plasmid stock to 90 µL of RNase-free water containing salmon sperm DNA (5 ng/mL) in a 1.5 mL microfuge tube (the 10⁻¹ dilution), then transfer 10 µL of that dilution to 90 µL of RNase-free water containing salmon sperm DNA (5 ng/mL) (the 10⁻² dilution), and repeat until the 10⁻⁸ dilution is reached

Viral RNA extraction from cell-free samples using the QIAamp viral RNA Mini kit

(Always follow the latest manufacturer’s instructions provided in the QIAamp viral RNA Mini kit — they prevail over the method’s instructions. Buffer AVL-carrier RNA is prepared fresh and is stable at 2 to 8°C for up to 48 hours. All centrifugation steps are carried out at room temperature, 23°C ± 3°C.)

Pipette 560 µL of prepared buffer AVL containing carrier RNA into a 1.5 mL microfuge tube

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Add 140 µL of each sample into the tube containing the buffer AVL-carrier RNA

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Pulse-vortex the microfuge tubes for 15 seconds

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Incubate the samples for 10 minutes at room temperature (23°C ± 3°C)

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Briefly centrifuge the microfuge tubes to remove drops from the inside of the lid

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Add 560 µL of non-denatured ethanol (96 to 99%) to the samples

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Pulse-vortex the microfuge tubes for 15 seconds

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Transfer 630 µL of the solution to a QIAamp Mini spin column (in a 2 mL collection tube) without wetting the rim

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Close the caps and centrifuge the spin columns at 6000 × g for 1 minute

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Transfer each spin column to a clean 2 mL collection tube and discard the previously used collection tubes

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Transfer the remaining 630 µL of each solution to its respective spin column and centrifuge at 6000 × g for 1 minute

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Repeat the transfer-to-clean-tube step

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Add 500 µL of buffer AW1 to each spin column, close the lids, and centrifuge at 6000 × g for 1 minute

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Transfer each spin column to a clean 2 mL collection tube and discard the previously used collection tube

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Add 500 µL of buffer AW2 to each spin column, close the lids, and centrifuge at 20000 × g for 3 minutes

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Place each spin column in a 1.5 mL microfuge tube and discard the previously used collection tubes

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Add 60 µL of buffer AVE to each spin column (alternatively, perform a double elution with two separate 30 µL applications of buffer AVE, each followed by centrifugation at 6000 × g for 1 minute)

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Close the caps and incubate at room temperature (23°C ± 3°C) for 1 minute, then centrifuge at 6000 × g for 1 minute

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Discard the spin columns and close the lids of the 1.5 mL microfuge tubes

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Add 20 units of RNase inhibitor (0.5 µL of RNaseOUT 40 U/µL) to the extracted RNA

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Use the RNA directly in RT-PCR or real-time RT-PCR, or store it at −70°C until needed

Conventional RT-PCR for the amplification of FCV RNA

Amplify RNA from each extracted RNA sample with the FCV primers CBK-1 and CBK-2 using the Qiagen OneStep RT-PCR kit (always follow the latest manufacturer’s instructions provided in the kit)

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Add 1.0 µL of each extracted RNA sample to 24.0 µL of RT-PCR reaction mixture (see the OneStep RT-PCR mix in section 6)

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Set up a negative control by adding 1.0 µL of RNase-free water to 24.0 µL of the same RT-PCR reaction mixture; for positive controls, add 1.0 µL of FCV RNA to 24.0 µL of the same mixture

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Insert the PCR tubes or 96-well plate in a thermal cycler and proceed with RT-PCR amplification according to the CBK cycling program in section 6

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After RT-PCR is completed, analyze the RT-PCR product by agarose gel electrophoresis (the agarose gel electrophoresis steps above) — if necessary, the amplicons can be stored at 4°C until analysis and/or confirmed by digestion with HpaII restriction endonuclease (the HpaII digestion steps above)

Agarose gel electrophoresis

Prepare a 2.0% (w/v) agarose gel in 0.5× TBE or 1× TAE buffer — dissolve the agarose by stirring on a hot plate or by microwaving for 1 to 2 minutes at high power until completely dissolved (clear liquid with no particles in suspension)

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Cool the agarose to around 45°C, then add concentrated ethidium bromide (EtBr) solution to obtain a final concentration of 0.5 µg/mL in the gel, mixing gently while avoiding bubble formation (alternatively, use SYBR Safe DNA gel stain at a final dilution of 1:10000; the addition of stain to the gel is optional if the gel is submerged in EtBr or SYBR Safe stain solution after migration)

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Pour into a gel tray without trapping bubbles, add a well-forming comb, and allow the gel to solidify for about 20 to 30 minutes

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Prepare the samples for electrophoresis — in clean microfuge tubes, mix 1.2 µL of 10× concentrated nucleic acid loading buffer into each microfuge tube containing 10 µL of RT-PCR product

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Remove the comb, place the tray with the gel in the electrophoresis apparatus, and fill the reservoir with 0.5× TBE or 1× TAE buffer to cover the gel to a depth of 4 mm; gently pipette the samples (approximately 11.2 µL) into the wells of the submerged gel, including a DNA molecular size marker (e.g. 100 bp DNA ladder) as well as positive, negative and reagent controls

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Connect the apparatus to the power supply with the cathode (−, black) at the top (near the sample wells) and the anode (+, red) at the bottom; apply approximately 100 volts and run for about 30 minutes, or until the tracking dye has spread about two-thirds of the length of the gel (voltage and time may be modified according to the size of the electrophoresis device and the length of the gel)

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Remove the gel from the tray and visualize the DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator — photograph the gels on Polaroid 667 film or with a digital processing system for analysis and record keeping (if stain was not added to the gel, stain it after migration by placing it in ethidium bromide solution (10 µg/mL) (the source prints “10 g/ml”) or SYBR Safe 1× solution for 15 minutes, rinse briefly with tap water, and visualize)

Reading conventional RT-PCR results

The amplicons generated by the FCV CBK-1 and CBK-2 primers are double-stranded DNA fragments of 218 bp — a positive PCR test yields a band specific to the targeted gene sequence, visible as an intense band on an EtBr/SYBR Safe stained agarose gel, its size verified against the DNA molecular size marker run on the same gel

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A negative PCR test normally produces no visible DNA bands — any sample giving bands not corresponding to the expected amplicon (non-specific amplification products) is considered negative

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A specific band must appear for the targeted positive control — absence of the positive-control band invalidates the test and the samples are re-analyzed

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Any band corresponding to the positive control occurring in the negative control with the FCV primers indicates possible contamination of the RT-PCR reaction mixture — the whole batch is considered suspect and is discarded, and the samples are re-analyzed using a new reagent batch

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Any test sample showing a distinct band with the FCV primers, corresponding to its positive control, is considered a presumptive positive — the PCR product is confirmed by digestion with HpaII restriction endonuclease (the HpaII digestion steps above)

HpaII restriction endonuclease digestion

Add 10 µL of each amplicon and 10 µL of RNase-free water to separate 1.5 mL microfuge tubes

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

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Incubate overnight in a water bath or heat block at 37°C

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After the reaction is completed, analyze the digested PCR product by agarose gel electrophoresis (the agarose gel electrophoresis steps above) — the two DNA fragments generated by HpaII are 114 and 104 bp in molecular size; bands of 114 and 104 bp as compared to the DNA molecular size marker (e.g. 100 bp DNA ladder) run on the same gel are confirmed as FCV amplicons (the two fragments may appear co-migrating on a 2.0% (w/v) agarose gel)

Calculating RT-PCR units

Determine the highest FCV dilution (the sample-handling dilution steps above) showing a positive result (the conventional RT-PCR reading section above) — this is the end-point dilution

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Take the reciprocal of this dilution to give RT-PCR units per quantity used for the RT-PCR (i.e. 1.0 µL)

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To obtain a concentration per mL, multiply the concentration obtained by the factor that gives 1000 µL (1 mL) — for example, if 1.0 µL of RNA was used and 10⁻⁴ was the highest dilution showing a band, multiply 10⁴ by 1000 to give 1.0 × 10⁷ RT-PCR units per mL

RT-PCR units per mL = (reciprocal of the end-point dilution) × 1000

• Where, reciprocal of the end-point dilution is the value taken from the highest dilution showing a positive result, per 1.0 µL of RNA used in the RT-PCR
• Where, 1000 is the conversion factor from 1.0 µL to 1 mL

(Viral RNA titres are usually expressed as RT-PCR units per mL.)

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

Amplify RNA from each extracted RNA sample in duplicate with the FCV primers and TaqMan probe (SH-FCV3-Q-A, SH-FCV3-Q-1 and SH-FCV3-P) using the Stratagene Brilliant II QRT-PCR Core Reagent kit (or equivalent) — in triplicate, run a minimum of 5 to 6 points of the corresponding validated standard curve of known copy numbers (always follow the latest manufacturer’s instructions provided in the kit)

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Add 2.0 µL of each extracted RNA sample or clone used for the standard curve to 23.0 µL of real-time RT-PCR reaction mixture (see the Brilliant II mix in section 6)

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Set up reaction controls in triplicate — for the No Template Control (NTC), add 2.0 µL of RNase-free water to 23.0 µL of the same mixture; as positive control, add 2.0 µL of previously confirmed FCV RNA or cDNA from the corresponding clone to 23.0 µL of the same mixture (in a two-step RT-PCR reaction, a no-RT control may be included by omitting the reverse transcriptase enzyme — it is expected to generate no real-time PCR signal)

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Insert the PCR strip tubes or 96-well plate in the spectrofluorometric thermal cycler and proceed with amplification according to the SH-FCV3 cycling program in section 6, taking fluorescence readings at the annealing/extension temperature (60°C)

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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

Reading real-time RT-PCR results

Results are displayed in an amplification plot reflecting the change in fluorescence during cycling — a positive real-time RT-PCR test generates a Ct (threshold cycle) value, defined as the cycle at which the fluorescence is statistically significant above the background signal of the probe; as PCR proceeds, the fluorophore produces fluorescent light (degradation of the TaqMan probe) over the background fluorescence of the linear probe quench — check for any abnormalities in the amplification plot

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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 fall between 15 and 35; a plot of Ct versus the logarithm of the copy number gives a straight line; an efficient real-time PCR assay has a slope of −3.3, an intercept of around 38, and an R² greater than 0.98; the amplification efficiency should be between 90 and 110% (optimal standard curves are as close to 100% as possible)

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A negative real-time RT-PCR test normally produces no fluorescence over the background signal of the probe for the entire run — it is validated with the positive control

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At a specific previously validated Ct, the fluorescence from the positive control should increase over the background fluorescence — absence of fluorescence in the positive control invalidates the test and the samples are re-analyzed

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Any Ct occurring in the negative control with the FCV primers and probe set indicates possible contamination of the real-time RT-PCR reaction mixture — the whole batch is considered suspect and is discarded, and the samples are re-analyzed using a new reagent batch

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Any sample showing a Ct with the FCV primers and probe set when the NTC values are negative, or when the NTC Ct is at least 5 cycles from the highest Ct value of the samples analyzed, is considered positive

Calculating genomic equivalent units

Confirm that the unknown sample’s Ct value falls within the standard curve, then estimate the initial genomic equivalent value of the cDNA from the mean of the duplicate Ct values of the sample compared to the standard curve

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To obtain the initial concentration in genomic equivalents per µL, divide the concentration obtained by the volume of RNA used as template — for example, if 2.0 µL of RNA were used and the mean Ct value corresponds to 4.0 × 10⁶ genomic equivalents on the standard curve, divide 4.0 × 10⁶ genomic equivalents by 2 µL, giving 2.0 × 10⁶ genomic equivalents per µL

Genomic equivalents per µL = (genomic equivalents from the standard curve) ÷ (µL of RNA used as template)

• Where, genomic equivalents from the standard curve is the value corresponding to the mean of the duplicate Ct values
• Where, µL of RNA used as template is the volume of extracted RNA added to the real-time RT-PCR reaction

(Viral RNA concentrations are usually expressed as genomic equivalent per µL of RNA used as template; using appropriate calculations, they can be expressed as genomic equivalent per unit of sample tested.)

5. Preparation of the cDNA clone for the real-time RT-PCR standard curve

Production of the cDNA fragment using conventional RT-PCR

Amplify FCV RNA with the real-time FCV primers (SH-FCV3-Q-A and SH-FCV3-Q-1) using the Qiagen OneStep RT-PCR kit with the RT-PCR CBK cycling program in section 6

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Add 2.0 µL of FCV RNA sample to 48.0 µL of conventional RT-PCR reaction mixture (double the OneStep RT-PCR mix recipe in section 6)

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After RT-PCR is completed, analyze the RT-PCR product by agarose gel electrophoresis (the agarose gel electrophoresis steps above) on a 3.0% (w/v) agarose gel — if necessary, the amplicons can be stored at 4°C (the amplicon generated by the SH-FCV3-Q-A and SH-FCV3-Q-1 primers is a double-stranded DNA fragment of 85 bp)

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Prepare the sample for electrophoresis — in a clean microfuge tube, mix 4.0 µL of 10× concentrated nucleic acid loading buffer and 40 µL of RT-PCR product

Purification of the RT-PCR product using the QIAquick gel extraction kit

(All reagents in this section are provided in the kit, except ethanol 96 to 99%. Add ethanol (96 to 99%) to Buffer PE before use. Always follow the latest manufacturer’s instructions provided in the kit.)

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel, minimizing the size of the gel slice by removing extra agarose

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Weigh the gel slice in a colorless microtube and add 6 volumes of Buffer QG to 1 volume of gel (100 mg ≈ 100 µL)

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Incubate at 50°C for 10 minutes or until the gel slice has completely dissolved, mixing by vortexing the tube every 2 to 3 minutes during the incubation (for 3% gels, a longer incubation may be necessary — solubilize the agarose completely)

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After the gel slice has dissolved completely, check that the color of the mixture is yellow (like Buffer QG without dissolved agarose) — if the color is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix until the mixture turns yellow (DNA binds efficiently to the QIAquick membrane only at pH ≤ 7.5)

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Add 1 gel volume of isopropanol to the sample and mix

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Place a QIAquick spin column in a provided 2 mL collection tube and apply the sample to the column to bind the DNA, then centrifuge for 1 minute at 17,900 × 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 µL — for larger volumes, load and spin again)

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Discard the flow-through and place the QIAquick column back in the same collection tube

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Add 0.5 mL of Buffer QG to the QIAquick column and centrifuge for 1 minute at 17,900 × g (13,000 rpm)

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To wash, add 0.75 mL of Buffer PE to the QIAquick column and centrifuge for 1 minute at 17,900 × g (13,000 rpm)

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Discard the flow-through and centrifuge the QIAquick column for an additional 1 minute at 17,900 × g (13,000 rpm) (residual ethanol from Buffer PE is not completely removed unless the flow-through is discarded before this additional centrifugation)

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Place the QIAquick column into a clean 1.5 mL microcentrifuge tube and elute the DNA by adding 50 µL of Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0 to 8.5) to the center of the QIAquick membrane — let stand for 1 minute, then centrifuge the column for 1 minute at 17,900 × g (13,000 rpm) (dispense the elution buffer directly onto the membrane for complete elution; the average eluate volume is 48 µL from 50 µL of elution buffer; when using water, make sure the pH is within range, and store the DNA at −20°C since DNA may degrade without a buffering agent; the purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but EDTA may inhibit subsequent enzymatic reactions)

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Verify the purified DNA by agarose gel electrophoresis (the agarose gel electrophoresis steps above), loading 9 µL of purified product and 1 µL of 10× concentrated nucleic acid loading buffer on a 3.0% (w/v) agarose gel

Cloning reaction with the TOPO TA Cloning kit and transformation of competent cells

(All reagents in this section are provided in the kit, except LB medium and LB agar plates containing the appropriate antibiotic and X-Gal. Always follow the latest manufacturer’s instructions provided in the kit. Transformation may be performed chemically or by electroporation — refer to the Invitrogen user manual for chemical transformation.)

Warm the vial of S.O.C medium to room temperature (23°C ± 3°C)

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Warm the selective plates (LB plates containing 50 µg/mL of kanamycin) at 37°C for 30 minutes

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Spread 40 µL of 40 mg/mL X-Gal in dimethylformamide (DMF) on each LB agar plate and incubate at 37°C until ready for use

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Place the electroporation cuvettes on ice and thaw on ice 1 vial of One Shot TOP10 Electrocompetent E. coli cells

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Dilute the salt solution 4-fold by adding 15 µL of water to 5 µL of salt solution

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Set up the TOPO cloning reaction by adding 4 µL of purified PCR product to 1 µL of diluted salt solution and 1 µL of pCR2.1-TOPO vector

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Mix gently and incubate for 5 minutes at room temperature, then place the reaction on ice

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Add 2 µL of the TOPO cloning reaction into a vial of One Shot TOP10 Electrocompetent E. coli cells and mix gently (do not mix by pipetting up and down)

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Carefully transfer the solution to a 0.1 cm cuvette, avoiding bubble formation

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Electroporate the sample using the electroporator’s own protocol

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Immediately add 250 µL of room-temperature S.O.C medium

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Transfer the solution to a 15 mL snap cap tube and shake for at least 1 hour at 37°C to allow expression of the antibiotic resistance genes

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Spread 10 to 50 µL from each transformation on a pre-warmed LB plate containing X-Gal and 50 µg/mL kanamycin and incubate overnight at 37°C

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Take 10 white colonies and culture them overnight at 37°C in 5 mL of LB medium containing 50 µg/mL kanamycin

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

Isolation of plasmid DNA from E. coli using the NucleoSpin Plasmid kit

(All reagents in this section are provided in the kit, except ethanol 96 to 99% and 1.5 mL microcentrifuge tubes. Always follow the latest manufacturer’s instructions provided in the kit.)

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

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Add 96 to 99% non-denatured ethanol to Buffer A4 before use (the appropriate volume is printed on the Buffer A4 bottle)

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Pipette 1.5 mL of saturated E. coli culture and centrifuge at 11,000 × g for 30 seconds to pellet the bacterial cells

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Discard the supernatant

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Resuspend the pellet in 250 µL of Buffer A1 by vigorous vortexing (check Buffer A2 for precipitated SDS before use — if a white precipitate is visible, warm the buffer for several minutes at 30 to 40°C until the precipitate dissolves completely, then cool it to room temperature, 23°C ± 3°C)

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Add 250 µL of Buffer A2 to the suspension, mix gently by inverting the tube 6 to 8 times, and incubate at room temperature for 5 minutes (do not vortex, to avoid releasing contaminating chromosomal DNA into the suspension)

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Add 300 µL of Buffer A3 to the suspension and mix gently by inverting the tube 6 to 8 times

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Centrifuge the suspension at 11,000 × g in a microcentrifuge for 5 minutes (repeat if the supernatant is not clear)

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Place a NucleoSpin Plus column in a 2 mL collection tube and load the supernatant from the previous step into the spin column

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Centrifuge at 11,000 × g for 1 minute and discard the flow-through

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Wash the NucleoSpin column with 500 µL of Buffer AW

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Centrifuge at 11,000 × g for 1 minute and discard the flow-through

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Reinsert the NucleoSpin column in the 2 mL collection tube and add 600 µL of Buffer A4 (with ethanol) to the column

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Centrifuge at 11,000 × g for 1 minute and discard the flow-through

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Recentrifuge at 11,000 × g for 2 minutes to remove residual ethanol from the silica membrane inside the spin column

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Place the NucleoSpin column in a 1.5 mL or 2 mL microcentrifuge tube (not included with the kit), elute the DNA by adding 50 µL of Buffer AE (5 mM Tris-HCl, pH 8.5) to the column, and incubate 1 minute at room temperature (23°C ± 3°C)

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Centrifuge at 11,000 × g for 1 minute — the plasmid DNA is now collected in Buffer AE at the bottom of the tube

Confirmation of the presence of the FCV insert

Add 2.0 µL of purified DNA plasmid (the plasmid isolation steps above), 2.0 µL of restriction endonuclease buffer (provided with the restriction endonuclease), 15.0 µL of RNase/DNase-free water, and 1.0 µL of EcoRI (10 U/µL) restriction endonuclease in a microfuge tube

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Incubate overnight in a water bath or heat block at 37°C

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After the reaction is completed, prepare the sample for electrophoresis — in a clean microfuge tube, mix 10 µL of the digested plasmid product and 1 µL of 10× concentrated nucleic acid loading buffer; analyze by agarose gel electrophoresis (the agarose gel electrophoresis steps above) on a 3.0% (w/v) agarose gel (the two DNA fragments generated by EcoRI are 3921 bp (plasmid) and 85 bp (FCV cDNA) in molecular size — bands of 85 bp as compared to a DNA molecular size marker (e.g. 100 bp DNA ladder) run on the same gel are confirmed as FCV cDNA)

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Sequence the insert to confirm its integrity (M13 forward and reverse primers may be used for sequencing; DNA sequencing can usually be contracted out to a local university or a biotechnology firm)

Plasmid linearization for use as a standard

Add 35.0 µL of purified DNA plasmid (the plasmid isolation steps above), 12.0 µL of restriction endonuclease buffer (provided with the restriction endonuclease), 65.0 µL of RNase/DNase-free water, and 10.0 µL of BamHI (10 U/µL) restriction endonuclease in a microfuge tube

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Incubate 4 hours in a water bath or heat block at 37°C

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Inactivate the restriction endonuclease by incubating 20 minutes in a water bath or heat block at 55°C

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After the reaction is completed, prepare the sample for electrophoresis — in a clean microfuge tube, mix 2.0 µL of the digested plasmid product, 8.0 µL of RNase/DNase-free water, and 1 µL of 10× concentrated nucleic acid loading buffer; analyze by agarose gel electrophoresis (the agarose gel electrophoresis steps above) on a 2.0% (w/v) agarose gel (the DNA fragment generated by BamHI is 4016 bp (3931 bp plasmid + 85 bp FCV cDNA) in molecular size — only one band should appear on the gel to confirm linearization of the cDNA clone)

Standard curve preparation for real-time RT-PCR

Determine the concentration in g/µL of the linear DNA plasmid stock (the linearized plasmid prepared above) with a spectrophotometer

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Evaluate the molar mass — bp length (plasmid + FCV insert) × 649 g/mol (molar mass for 1 kb); (3931 bp + 85 bp) × 649 g/mol = 2.606 × 10⁶ g/mol

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Evaluate the copy number (genomic equivalents)/µL — copy number/µL = [(concentration of linearized plasmid, g/µL) ÷ (molar mass)] × (6.023 × 10²³); for example, if the concentration of linearized plasmid is 143.89 ng/µL (i.e. 1.439 × 10⁻⁷ g/µL): copy number/µL = [(1.439 × 10⁻⁷ g/µL) ÷ (2.606 × 10⁶ g/mol)] × (6.023 × 10²³) = 33.259 × 10⁹, or 3.326 × 10¹⁰

Copy number per µL = [(concentration of linearized plasmid) ÷ (molar mass)] × (6.023 × 10²³)

• Where, concentration of linearized plasmid is the value determined with the spectrophotometer, in g/µL
• Where, molar mass is the bp length (plasmid + FCV insert) × 649 g/mol, in g/mol
• Where, 6.023 × 10²³ is Avogadro’s number

↓

Dilute the linear plasmid stock in RNase/DNase-free water to obtain a concentration of 1 × 10⁹ copies (genomic equivalents)/µL

↓

Generate the standard curves as described in the sample-handling section above

6. Primer sequences and temperature cycling programs

RT-PCR primers (conventional RT-PCR, 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′

(Oligonucleotide primers can usually be synthesized under contract by a local university or a biotechnology firm; primers for the best results are purified by High Pressure/Performance Liquid Chromatography, HPLC.)

Temperature cycling program for the CBK primers

• Step 1 — reverse transcription: 30 minutes at 50°C
• Step 2 — initial PCR activation: 15 minutes at 95°C (the HotStarTaq DNA Polymerase is activated; the Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA template is denatured)
• Step 3 — 32 cycles of:
• Denaturation: 45 seconds at 94°C
• Annealing: 45 seconds at 53°C
• Extension: 45 seconds at 72°C
• Step 4 — final elongation: 10 minutes at 72°C

Qiagen OneStep RT-PCR kit mix (recipe for a batch equivalent to 20 reactions)

(All stock solutions are stored at −20°C until used. Always follow the latest manufacturer’s instructions provided in the kit. Work RNase-free — see the Precautions. Mixes are prepared with all the reagents on ice or on a refrigerated rack; 25 µL of RT-PCR mix in 200 µL tubes are used to reduce the cost of reagents.)

• RNase-free water: 280.0 µL for the batch, 14.0 µL per tube
• 5× Qiagen OneStep RT-PCR buffer (5×): 100.0 µL for the batch, 5.0 µL per tube (final 1×)
• dNTP mix (10 mM of each dNTP): 20.0 µL for the batch, 1.0 µL per tube (final 400 µM)
• CBK-1 primer (10 µM): 30.0 µL for the batch, 1.5 µL per tube (final 0.6 µM)
• CBK-2 primer (10 µM): 30.0 µL for the batch, 1.5 µL per tube (final 0.6 µM)
• Qiagen OneStep RT-PCR Enzyme Mix: 20.0 µL for the batch, 1.0 µL per tube
• Total volume: 480.0 µL for the batch — distribute 24.0 µL per tube
• Template per tube: 1.0 µL of extracted RNA

Temperature cycling program for the SH-FCV3 primer and probe system (real-time RT-PCR)

• Step 1 — reverse transcription: 30 minutes at 50°C
• Step 2 — initial PCR activation: 10 minutes at 95°C (the SureStart Taq DNA Polymerase is activated; the Reverse Transcriptase is inactivated and the cDNA template is denatured)
• Step 3 — 45 cycles of:
• Denaturation: 15 seconds at 95°C
• Annealing + extension: 60 seconds at 60°C (take the fluorescence reading at this temperature)

Stratagene Brilliant II QRT-PCR Core Reagent kit mix, OneStep (recipe for a batch equivalent to 102 reactions)

(All stock solutions are stored at −20°C until used. Always follow the latest manufacturer’s instructions provided in the kit. If ROX is used as a passive reference dye, dilute it 1:500 in nuclease-free PCR-grade water prior to use; if ROX is omitted, replace it with an equivalent volume of RNase-free water.)

• RNase-free water: 1364.25 µL for the batch, 13.375 µL per tube
• 10× Core RT-PCR buffer (10×): 255.0 µL for the batch, 2.5 µL per tube (final 1×)
• Magnesium chloride (50 mM): 255.0 µL for the batch, 2.5 µL per tube (final 5.0 mM)
• SH-FCV3-Q-A primer (10 µM): 76.5 µL for the batch, 0.75 µL per tube (final 0.3 µM)
• SH-FCV3-Q-1 primer (10 µM): 76.5 µL for the batch, 0.75 µL per tube (final 0.3 µM)
• SH-FCV3-P probe (10 µM): 51.0 µL for the batch, 0.5 µL per tube (final 0.2 µM)
• dNTP mix (20 mM; 5 mM of each dNTP): 102.0 µL for the batch, 1.0 µL per tube (final 800 µM)
• Reverse transcriptase: 102.0 µL for the batch, 1.0 µL per tube
• Reference dye (ROX) (1 mM stock, diluted to 2 µM): 38.25 µL for the batch, 0.375 µL per tube (final 0.03 µM)
• SureStart Taq DNA Polymerase (5 U/µL): 25.5 µL for the batch, 0.25 µL per tube (final 0.05 U/µL)
• Total volume: 2346.0 µL for the batch — distribute 23.0 µL per tube
• Template per tube: 2.0 µL of extracted RNA

Real-time RT-PCR primers and probe (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-FCV3-P (probe), 30 bases: 5′ – CGC CTT ACG GAT ATG AGC AGC CAC ATT AAC – 3′ (ordered as 5′ HEX (Hexachlorofluorescein) – 3′ IBQ (Iowa Black Quencher); other fluorochrome/quencher combinations are possible)

7. Limitations

• The method will not differentiate between infectious and non-infectious viruses
• The use of thermal cyclers other than the models stated, or of a reagent kit other than the specified kits, may alter the performance of the RT-PCR reactions — cycling parameters or reagent concentrations may need to be optimized for different models or kits

8. Precautions

• The laboratory supervisor must ensure that the analysis is completed in accordance with ISO/IEC 17025:2005 (or latest version), General requirements for the competence of testing and calibration laboratories
• 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
• Buffers AW2 and AVE contain sodium azide as a preservative, which is highly toxic — take appropriate safety measures and wear gloves when handling
• 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
• Great care should be taken when working with RNA to avoid contact with ubiquitous RNases — RNases are very stable and 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; bake glassware (cleaned, thoroughly rinsed) at above 240°C for four or more hours before use
• All reagents, DNase/RNase-free water, pipette tips and other materials coming into contact with samples or RT-PCR reagents should be sterile or autoclaved prior to use to remove DNases and other contaminants; prepare all reagents in a laminar flow cabinet that has never been exposed to FCV or FCV RT-PCR products
• Ethidium bromide is a potent mutagen — use nitrile gloves when handling; dispose of all solutions and used gels according to the waste-disposal guidelines
• UV light can cause eye damage — wear safety goggles
• Maintain block heaters or water baths 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

References

• Laboratory Procedure OPFLP-06, Detection of feline caliciviruses using the conventional and real-time reverse-transcriptase polymerase chain reaction, March 2010, Health Canada, Compendium of Analytical Methods, Volume 5
• Brilliant II QRT-PCR Core Reagent Kit, 1-Step Instruction Manual. 2006
• NucleoSpin Plasmid kits user Manual. March 2008
• QIAamp Viral RNA Mini Handbook. December 2007
• Qiagen OneStep RT-PCR Kit Handbook. February 2008
• QIAquick Spin Handbook. March 2008. pp. 25–26
• SYBR Safe DNA gel stain. July 2006
• TOPO TA Cloning user Manual. April 2006