Detection of Listeria monocytogenes by the Nucleic Acid Sequence-Based Amplification Technique
3.3. Detection of Amplicons
3.3.1. Detection of Amplicons from DNA Targets
InSubheading 3.2.1., target sequences originating from DNA molecules (e.g., genomic DNA) were amplified in the presence of digoxigenin-11-UTP, resulting in the incorporation of this label in the amplicons (predominantly minus-strand RNA). The procedure outlined in this section pertains to a microtiter plate-based assay for the detection of the digoxigenin-labeled NASBA amplicons by hybrid- ization with an immobilized DNA probe. The hybridized amplicons are then detected by sequential reactions of the microtiter plate wells with anti-digoxigenin antibody–peroxidase conjugate and tetramethylbenzidine substrate solution. The intensity of the resulting color in the wells is proportional to the amount of amplicon produced in the NASBA reaction.
The DNA probe-coated microtiter plates must be prepared in advance. In the present NASBA system, the DNA probe is conveniently made by amplification of a portion of the target sequence using the PCR technique.
Detecting L. monocytogenes with NASBA 77 3.3.1.1. PREPARATION OF DNA PROBE
A capture DNA probe can be prepared using the PCR technique with purified L. monocytogenes genomic DNA as template:
1. In a 0.6-mL-capacity microcentrifuge tube, combine 10 µL of distilled H2O containing 10 ng of L. monocytogenesgenomic DNA (seeSubheading 3.1.1.) with 89.5 µL of PCR reaction mixture (11 mMTris-HCl (pH 8.3); 0.22 mMeach of ATP, CTP, GTP, and TTP; 2.2 mM MgCl2; 55 mM KCl; and 0.11% (v/v) Triton X-100) containing 1.1 µM each of primers P1 and P2 (see Subheading 2.3.1.1.). Overlay the mixture with mineral oil, then place in a thermal cycler set to hold the temperature at 80°C for for 10 min.
2. Add 0.5 µL containing 1 U Taq DNA polymerase to the tube, then subject the sample to 35 cycles of the following sequence: denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s and primer extension at 72°C for 90 s. Allow an additional 2 min at 72°C after the last cycle in order to ensure completion of primer extension.
3. Gently insert a pipet tip under the mineral oil layer and remove all of the PCR reaction mixture to a new 1.5-mL-capacity microcentrifuge tube. Add 0.1 vol of 3 M ammonium acetate (pH 5.2), followed by 2.5 vol of ethanol, and place at –20°C for at least 2 h.
4. Proceed as for steps 18–21 in Subheading 3.1.1.
3.3.1.2. IMMOBILIZATION OF DNA PROBE ON A MICROTITER PLATE
1. Denature an aliquot of DNA probe stock by heating at 100°C for 10 min, then immediately dilute to a final concentration of 2 µg/mL in ice-cold coating buffer (0.3M Tris-HCl (ph 8.0); 0.5 M MgCl2; 1.5 M NaCl).
2. Transfer 100 µL of diluted, denatured DNA to each well of a microtiter plate, then seal the wells (e.g., using adhesive tape or microtiter plate sealing film pressed firmly over the wells), and incubate at 37°C for 16 h.
3. Empty the wells of their contents and allow to air dry. The DNA can be crosslinked to the plastic by exposure to ultraviolet light (254 nm) for 3 min.
4. Wash the wells three times with approx 200 µL wash buffer (0.1 M Tris-HCl (pH 8.0); 2 mMMgCl2; 1 MNaCl; 0.1% (v/v) Tween-20). Note that the wells can be washed by simply filling each with wash buffer using a squirt bottle, and empty- ing by vigorously shaking the plate upside down. Ensure that no residual buffer remains in the wells.
5. Block the wells by incubation at 37°C for 1 h with 100 µL of hybridization solution (5X SSC [1X SSC is 0.15 MNaCl plus 0.015 Msodium citrate]; 1 % (w/v) block- ing reagent; 0.1 % (w/v) N-lauroylsarcosine; 0.02 % (w/v) sodium dodecyl sulfate;
50 % (v/v) formamide). Wash the wells three times with PBST (0.01 Mphosphate (ph 7.2), 0.15 MNaCl; and 0.05 % (v/v) Tween-20). At this point, the wells can be emptied and air dried for storage at 4°C for a maximum of 4–6 wk.
78 Blais and Turner 3.3.1.3. ASSAY OF NASBA AMPLICONS
1. In a DNA probe-coated microtiter plate well, combine 25 µL of hybridization solution (seeSubheading 3.3.1.2.) with 25 µL of NASBA reaction product (see Subheading 3.2.1.) , and incubate at 56°C for 90 min.
2. Wash the wells with PBST (seeSubheading 3.3.1.2.), and incubate at room tem- perature for 20 min with 100 µL of anti-digoxigenin antibody–peroxidase conju- gate diluted 1:2 000 in PBST containing 0.5 % (w/v) blocking reagent (PBST-B).
3. Wash the wells with PBST as before, and incubate at room temperature for 20 min with 100 µL of tetramethylbenzidine microwell substrate solution. A blue color will develop in the wells in the presence of bound peroxidase (positive samples).
Stop the reaction by the addition of 50 µL per well of 1 M H2SO4 (this will change the color from blue to yellow).
4. Using a microtiter plate reader, measure the absorbance in the wells at 450 nm (A450). Generally, negative control samples in which no template was added to the NASBA reaction mixture should give A450values below 0.10, whereas any absorbance value above this level should be considered as a positive result.
However, the cutoff absorbance values for different systems will have to be determined empirically. Alternatively, if a microtiter plate reader is not avail- able, reactions can be scored qualitatively by visual examination of the wells.
3.3.2. Detection of Amplicons from RNA Targets
In Subheading 3.2.2., target sequences originating from RNA molecules (i.e., mRNA) were amplified in the presence of biotin-11-UTP, in order to incorporate a biotin label in the amplicons. This section describes a simple membrane dot-blot technique as an alternative to the microtiter plate-based method for the detection of NASBA amplicons. In this approach, an oligonucleotide probe complementary to a portion of the NASBA amplicon (minus-strand RNA) is immobilized as a spot in the center of a small nylon membrane disc. For the assay, the disc is incubated with NASBA reaction product, and the amplicon is captured by hybridization with the immobilized probe at the center of the disc. The bound amplicon is then detected by sequential reactions with a streptavidin peroxidase conjugate and tetramethylbenzidine substrate solu- tion. In this manner, positive results are visualized on the basis of color formation (i.e., a blue spot) in the center of the disc.
3.3.2.1. PREPARATION OF PROBE-COATED DISCS
1. Dissolve the 56-mer oligonucleotide probe (seeSubheading 2.3.2.1.) at a final concentration of 30 ng/µL in 6 X SSC (seeSubheading 3.3.1.2.), and apply 1 µL to the center of an approx 8-mm nylon membrane disc (nylon membrane discs can be punched from a sheet of membrane using a clean standard hole puncher). Allow the disc to air dry, then expose to ultraviolet light (254 nm) for 5 min to crosslink
Detecting L. monocytogenes with NASBA 79 the probe to the membrane. Mark the edge of the disc with a pencil to keep track of the side receiving the DNA spot (you can also scribe a number or letter to permit sample identification later).
2. Block the disc by incubation at 52°C for 1 h in hybridization solution (see Subheading 3.3.1.2.) containing 25% (v/v) formamide instead of 50% (v/v). Note that several discs can blocked together in a bulk volume of hybridization solution (i.e., sufficient volume to submerge all of the discs).
3. Wash disc three times for 5 min at room temperature with PBST in a 250-mL- capacity beaker (constant gentle agitation). Note that several discs can be washed together in a bulk volume (e.g., 100 mL) of PBST. At this stage, discs can be used immediately in the dot blot assay or stored dry at 4°C.
3.3.2.2. DOT-BLOT ASSAY
1. Place a probe-coated disc in a 1.5-mL-capicity microcentrifuge tube containing 0.5 ml of hybridization solution, then add 25 µL of NASBA reaction product (see Subheading 3.2.2.1.) and mix well. Place the tube in a water bath adjusted to 52°C, and incubate for 30 min.
2. Drain the liquid (if necessary, touch lightly with the corner of a paper towel or blotting paper to draw off residual liquid) and, leaving the disc in the tube, wash three times for 5 min at room temperature with 1.5 mL of PBST (vortex briefly after addition of PBST).
3. Leaving the disc in the tube, add 0.5 mL of streptavidin–peroxidase conjugate diluted to 0.25 µg/mL in PBST containing 0.05% blocking reagent, and incubate at room temperature for 15 min.
4. Wash disc with PBST as before, then remove from tube and place on the flat surface of a Petri dish with the side bearing the DNA spot facing upward. Pipet 50 µL of tetramethylbenzidine membrane peroxidase substrate solution on the disc, and incubate at room temperature for 15 min. Reactions on the discs are scored qualitatively as follows: positive, colored spot formation at center of disc;
negative, no colored spot formation.
4. Notes
4.1. Selection of Oligonucleotides