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

154 Olmos et al.
This nested RT-PCR protocol coupled with a preparation of squashed or printed
samples on paper (10) would allow the detection of RNA targets from a number of
viruses in individual insect vectors, as well as in plant materials, animal fluids, or
tissues. The increased sensitivity provided with this method permits the amplification of
RNA targets from individual viruliferous aphids carrying stylet-borne (nonpersistent)
and semipersistent plant viruses (11,12) without the need of a preliminary purification
of nucleic acids.
Conditions for multiplex nested RT-PCR can be easily established using the device
described in Fig. 2. The simultaneous amplification of four RNA viruses and a
bacterium from olive trees by multiplex nested RT-PCR without interference among
primers has been successfully achieved in our laboratory (23). This system can include
the use, if necessary (see Note 2), of an immunocapture (IC) phase in the same tube
(IC-nested RT-PCR) (12). Recently, a new technique (called co-operational PCR/
Co-PCR) for amplification of nucleic acids targets, based on a simple tetraprimer
reaction has been described, with a sensitivity similiar to nested RT-PCR (24).
2. Materials
1. Eppendorf tubes (see Notes 2 and 3) (Cultek, Thermowell tubes cat no: 6530).
2. Sterile 200-µL pipet tip cones (see Fig. 2 and Notes 1 and 4)(Daslab, cat. no: 16-2001).
3. 10× RT-PCR buffer: 500 mM KCl, 100 mM Tris-HCl (pH 9.0 at 25°C), 1% Triton X-100
(supplied with Taq DNA polymerase).
4. Triton X-100 (Merck, Art. 8603).
5. DMSO (Sigma, cat. no. D8418).
6. MgCl 2 (25 mM; supplied with Taq DNA polymerase).
7. dNTPs (5 mM, Pharmacia, cat. no. 27-2035-02).
8. AMV reverse transcriptase (Pharmacia, cat. no. M5108).
9. Taq DNA polymerase (Promega, cat. no. M1865).
10. Primers for citrus tristeza virus (CTV) detection (see Subheading 3.2., step 1a):
External primers (100 µM)
• PEX1 (5′ TAA ACA ACA CAC ACT CTA AGG 3′)
• PEX2 (5′ CAT CTG ATT GAA GTG GAC 3′)
Internal primers (100 µM)
• PIN1 (5′ GGT TCA CGC ATA CGT TAA GCC TCA CTT 3′)
Primers for plum pox virus (PPV) detection (see Subheading 3.2., step 1b):
External primers (100 µM
• P10 (5′ GAG AAA AGG ATG CTA ACA GGA 3′)
• P20 (5′ AAA GCA TAC ATG CCA AGG TA 3′)
Internal primers (100 µM)
• P1 (5′ ACC GAG ACC ACT ACA CTC CC 3′)
• P2 (5′ CAG ACT ACA GCC TCG CCA GA 3′)
11. Micropipets (Gilson, pipetman P10, P20, P100, P200).
12. Microfuge (Heraeus, Biofuge PICO).
13. Thermal cycling machine with a heated lid (Techne, PHC-3).
14. Electrophoresis system (Bio–Rad, sub-cell gt/powerpac 300 power supply system Cat.
No. 165-4350).
15. Ethidium bromide (10 µg/mL in water; AppliChem, A1152,0025).
16. Transilluminator (TDI, Sepctroline Model TC-312A 312 nm UV).