1. To increase the speed of the process, several scalpels were used in rotation. The scalpel blades were decontaminated by successive washes of 1 min in duration in the following solutions; water, 0.2 M NaOH, water, 96% ethanol, and then briefly flamed to remove the ethanol.
2. Potatoes are sampled on a disposable surface (such as a polythene bag) on top of a ceramic tile, giving a clean cutting surface for each tuber.
3. For 150 samples, 2X 1 mL of enzyme solutions were made in 1.5-mL microfuge tubes and mixed on a culture wheel at room temperature until dissolved (the enzyme solution should be made in advance as it takes some time to dissolve). Before the addition to the tuber extract, 2 mL of the enzyme solution was mixed with 8 mL of SEB in a pipet trough, to give the correct dilution.
4. To allow for inaccuracies in pipetting, the master mixes for the RT step, and subsequently for PCR, should be prepared for more than
Fig. 1. IC-RT-PCR of potato latent virus. Agarose gel analysis of IC-RT-PCR assays from infected potato plants, immunocaptured using a PVS° polyclonal antibody, which also had affinity to potato latent virus (10). Lane M 100-bp molecular weight standard (SuperLadder-Low; ABgene); lane 1, negative control (water); lane 2, positive control (potato virus S); and lane 3, potato latent virus.
the actual number required. As an example, a master mix sufficient for one hundred samples should be prepared for use in for 96 reactions.
5. Because of the sensitivity of PCR assays, it is most important to minimise the potential danger of cross-contamination. Ideally, PCR laboratories should consist of two separate rooms, each containing their own equipment (e.g., pipetors). One room should be dedicated to the setting up of RT reactions and PCR assays only. Both laboratories should use aerosol-resistant tips to prevent carry-over of sample within the barrel of the pipet. Post-PCR tubes should never be opened in the room used to set up PCRs as this is probably the most potent source for potential contamination. A laminar-flow cabinet, particularly one designed for PCR, should be considered a requirement for a PCR room in situations where a large number of samples will be processed.
6. Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse tran-scriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the SuperScript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically.
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