(i) Sample preparation
1) For best results, isolates should be grown in RD monolayer cultures in Minimal Essential Medium (MEM) (lacking fetal calf serum) to produce high-titre stocks. Since poliovirus grows to higher titres in RD cells, as compared to L20B cells, the additional virus in the RD passage will give a stronger signal in the probe hybridization test. Large amounts of fetal calf serum in the virus sample can reduce the binding of the RNA to the membrane resulting in a weaker signal in the test.
Caution: Steps 2 to 7 should be performed in a biological safety cabinet.
2) After development of 4+ CPE, suspend the infected cells, and liberate virus by two freeze-thaw (dry ice-ethanol bath or freezer/36°C) cycles.
3) Prepare “formaldehyde mix” (avoid breathing formaldehyde fumes, use a fume hood if available):
20X SSC
0.3 ml/sample
37% formaldehyde 0.2 ml/sample
(20X SSC is 3.0 M NaCl, 300 mM sodium citrate; pH 7.0.)
4) Dispense 0.5 ml formaldehyde mix into each 1.5 ml microcentrifuge tube.
5) Transfer 0.6 ml of culture fluid to a separate 1.5 ml tube. Pellet the cellular debris in a microcentrifuge (>10 000 g for 1 minute).
6) Transfer 0.5 ml of supernatant to the appropriate 1.5 ml tube containing the formaldehyde mix.
7) Vortex thoroughly to mix and incubate at 65°C for 15 min.
Note: Incubation at 65°C in 3.7% formaldehyde completely inactivates poliovirus infectivity. Subsequent work can be performed on an open laboratory bench.
Because RNA molecules are degraded upon prolonged exposure to formaldehyde, prepare only enough formaldehyde-treated samples as will be used immediately.
(ii) Immobilization of poliovirus RNA onto membrane filters
1) Cut the corners from the sheets of the thicker filter paper and the nylon membrane filters so that the alignment pins in the manifold will be clear. Mark the corner of the nylon membrane with a pencil to identify well A1. Do not touch the nylon filters with your fingers (wear gloves and use filter forceps), as oils from the skin will interfere with RNA binding to the filter.
2) Saturate a piece of thick filter paper with 20X SSC and place it on the manifold.
3) Float the nylon membrane in a shallow container of distilled water to wet. After 30 seconds invert the membrane to thoroughly wet it.
4) Drain off excess water and briefly soak the membrane in 20X SSC by the method described in step 3.
5) Position the nylon membrane on top of the thicker filter paper. Clamp the “sandwich” together and gently apply vacuum. To ensure that none of the wells is blocked, filter 200 ml of 6X SSC through all wells. Air bubbles prevent complete filtration of the sample and can be removed by gently tapping the manifold on the bench. After the wells are cleared, turn off vacuum.
6) Apply samples (up to 0.2 ml) to the nylon membrane with a micropipette by dispensing down the wall of each well.
7) Spot samples in four identical sets, one set for each probe to be used. Positive and negative control RNA transcripts for each serotype should be included in each test run. At least two sets are needed:
- one set for the group probe, and
- one set for each strain-specific probe.
8) After all samples have been dispensed, gently apply vacuum until all wells are empty. Disassemble the manifold and carefully remove the nylon membrane.
9) Irradiate the nylon membrane on a transilluminator for three minutes to cross-link the RNA to the nylon membrane. Caution: avoid exposure to ultraviolet radiation. Wear a face shield, long sleeve lab coat, and plastic gloves. The nylon filter is now ready for hybridization.
(iii) Hybridization
1) Pre-hybridization step: Cut the nylon membrane into separate sample sets. Place nylon membranes in plastic sandwich boxes containing 1 ml of hybridization buffer [6x SSC, 50% (v/v) formamide, 0.1% (w/v) SDS, 2% (v/v) blocking reagent] for each cm2 of membrane. Several filters may be placed in the same box. Seal plastic boxes and incubate at 65°C for two hours in a shaking water bath (120 RPM).
2) Hybridization step: After pre-hybridization, drain the buffer and add 0.1 ml of hybridization buffer for each cm2 of nylon membrane (one-tenth the volume used for pre-hybridization. For example, for 24 dots use 5 ml probe in 5 ml buffer).
3) Add the probe to the hybridization buffer using a micropipette.
4) Seal each box and transfer to a 65°C shaking water bath (120 RPM).
5) Incubate overnight.
6) Transfer the nylon membranes to shallow containers, such as sealable plastic food containers, for washing. Hybridization solution containing probes can be stored at -20°C for reuse up to three times.
(iv) Filter washing
1) Wash the nylon membranes once for 15 minutes at 75°C with shaking in 25 ml Wash Buffer 1 (2X SSC, 0.1% (w/v) sodium dodecyl sulfate [SDS]).
2) Wash the nylon membranes once for 15 minutes at 75°C with shaking in 25 ml Wash Buffer 2 (0.1X SSC, 0.1% (w/v) SDS).
(v) Binding of anti-DIG Fab alkaline phosphatase conjugates to bound RNA probes
1) Following hybridization and post-hybridization, equilibrate the membrane in 25 ml Buffer A (100 mM maleic acid, 150 mM NaCl; pH 7.5) for 1 min.
2) Decant and drain Buffer A. Add 25 ml Buffer B (2% blocking reagent in Buffer A). Do not allow the membrane to dry.
3) Incubate filter for at least one hour with gentle shaking.
4) Near the end of the blocking step, dilute the anti-DIG Fab alkaline phosphatase conjugate 1:5000 in Buffer B for a working concentration of 150 mU/ml.
5) Remove the membrane from Buffer B (step 3) and transfer it to the Fab conjugate solution.
6) Incubate for 30 minutes with gentle shaking.
7) Drain the conjugate solution and gently wash the membrane twice for 15 minutes each in 20 ml of Buffer A.
8) Equilibrate the washed nylon membrane in 10 ml freshly prepared Buffer C (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2; pH 9.5) for 2 minutes.
(vi) Hybrid detection using chromogenic substrates
1) Prepare chromogenic substrate (45 ml of 100 mg/ml NBT and 35 ml of 50 mg/ml BCIP in 10 ml Buffer C). Typically 10 ml is required for each 100 cm2 of membrane. NBT is 4-nitroblue tetrazolium (stock in 100% dimethyl formamide [DMF]). BCIP is 5-bromo-4-chloro-3-indolyl-phosphate (stock in 70% DMF). Store NBT and DMF in dark in glass or polypropylene tubes.
2) Drain Buffer C. Add chromogenic substrate.
3) Incubate for 30 minutes in dark. If satisfactory colour development is observed, rinse filter in Buffer C.
4) Record results by Polaroid photography.