John M. S. Bartlett.pdf - Bio-Nica.info

156 Olmos et al.
Table 2
Volume and Concentration of Reactives for PPV Detection by Nested RT-PCR
Ingredients Cocktail A (RT-PCR) Cocktail B (nested-PCR)
10X RT-PCR buffer to 13.00 µl to 11.00 µl
MgCl 2 (25 mM) to 13.60 µl –
dNTPs (5 mM each) to 12.25 µl –
Triton ® X-100 (4%) to 12.50 µl –
3′ external primer (1 µM) to 13.00 µl 3′ internal primer (100 µM) to 10.80 µl
(Pe1 in Fig. 1) (Pi1 in Fig. 1)
5′ external primer (1 µM) to 13.00 µl 5′ internal primer (100 µM) to 10.80 µl
(Pe2 in Fig. 1) (Pi2 in Fig. 1)
DMSO to 11.50 µl –
H 2 O to 30.00 µl to 10.00 µl
AMV (10 U/µl) to 10.20 µl –
Taq Pol (5 U/µl) to 10.20 µl –
4. RT-PCR (see Note 6): 42°C for 45 min (reverse transcription), 94°C for 2 min (denaturation
and reverse transcriptase inactivation), 20 to 25 cycles (92°C for 30 s [denaturation], 45 or
50°C (see Subheading 3.2., step 1) for 30 s [annealing], and 72°C for 1 min [extension],
and final elongation at 72°C for 10 min.
5. Vortex the Eppendorf tube and centrifuge (pulse at 6000g for 2 s) to mix the second
cocktail with the RT-PCR products.
6. Nested-PCR (see Notes 7–9): 35 to 40 cycles (92°C for 30 s [denaturation], 60°C [see
Subheading 3.2., step 1] for 30 s [annealing], and 72°C for 1 min [extension] and final
elongation at 72°C for 10 min.
7. Electrophoresis (see Fig. 3): Load 10 µL of amplification products onto a 3% agarose
gel in 0.5× TAE and perform electrophoresis at 100 V for 30 min. Stain the agarose
gel with ethidium bromide (0.5 µg/mL) for 15 min and visualize the amplicons under
ultraviolet light.
4. Notes
1. The nested and heminested RT-PCR in a single closed tube described in this protocol
is based on the use of a simple device (see Fig. 2). Other patented compartmentalized
Eppendorf tubes with pockets could be commercialized by the industry in the near future,
consequently avoiding the need to prepare cones. Accidental flow of the second PCR mix
from the tip device might be caused by an incorrect manipulation of the device or by the
use of pipet tips wider than standard. This can be solved by closing (heating) the tip end.
In this case, after the RT-PCR it will be necessary to invert the tube, vortex to mix the
reagents, and centrifuge to collect all components in the bottom of the tube.
2. A previous capture step (immunocapture or print capture) improves the detection of some
RNA targets by RT-PCR. Conventional immunocapture is usually performed by pre-coating
Eppendorf tubes with 100 µL of carbonate buffer (pH 9.6) containing immunoglobulins
(2 µg/mL) of a known specificity (6–8). Normal immunoglobulins from nonimmunized
rabbits, bovine serum albumin, or skimmed milk can be also successfully used in this capture
phase. However, because of the high sensitivity of nested or heminested RT-PCR, this step
can usually be omitted. In this case, the detection of RNA targets can be performed by direct
incubation of 100 µL of the sample (crude extract or tissue print Triton X-100 extracts from