Avoiding false positives in PCR-based identification methods for nonsterile plant pathogens

Molecular approaches to species-specific identification are frequently applied in diagnostic, epidemiological, and phylogenetic studies. Many of these methodologies, such as polymerase chain reaction (PCR), random amplified polymorphic DNA, and restriction-fragment length polymorphism assays, make use of universal-primer sequences during the development of the assays or even in routine applications. This practise, particularly with environmental samples, can lead to erroneous conclusions, because samples may be contaminated with a wide range of diverse DNA sequences. As an example, in the course of developing PCR-based diagnostics for common nematode plant pathogens, in this study, the 18S-25S intragenic regions from genes encoding rRNA in the target organisms were isolated by PCR amplification. Employing "universal" primers, products of various sizes were amplified from DNA in six different pathogenic species, which had been isolated from the field and identified morphologically. While the observed size heterogeneity was very promising with respect to molecular identification, subsequent sequence analyses revealed several examples of contaminating DNA. Some reactions with universal primers did amplify rDNA sequences from the target nematodes but other reactions preferentially amplified rDNA sequences from small amounts of contaminating organisms, which were associated with the nematode samples. These results illustrate the necessity for sequence analyses when product size differences are adopted to identify closely related organisms and sampling cannot be carried out under sterile conditions.