Problems Encountered with Nested PCR to Diagnosis of Phytoplasmas

Introduction: Phytoplasmas are phytopathogenic bacteria without cell walls that can be found in plant phloem, have been found associated with numerous diseases of plants worldwide. A sensitive and precise diagnostic test for detection of phytoplasma-infected plant is critical to avoid using infected planting material and dispersal of these agents. The detection of phytoplasma from plant tissues by PCR, requires using DNA extraction methods that extract high level phytoplasmas DNA with less plant inhibitors. Usually Phytoplasma diagnostics have been based on the 16S rRNA gene and the 16S–23S rRNA spacer region because universal primers that design for replication these regions could detect different groups of phytoplasmas but diagnostics based on these primers can be problematic, with occasional false positives, through amplification of some bacterial genomes that might be present in a plant sample. These primers also have sequence homology to chloroplasts and plastids and increase the risk of false positives so it is important to guard against false negatives and positive during such detection techniques. Materials and Methods: Health and infected Lime samples with Candidatus phytoplasma aurantifolia were used respectively as negative and positive samples. DNA was extracted by the CTAB methods, SDS method, Fermentas DNA extraction kit, column based Method (Genet Bio genomic DNA isolation kit) and compared to remove inhibitors and reduced false negative reactions in nested PCR detection method. DNA samples were tested for phytoplasma infection by direct PCR using the universal phytoplasma primer pair P1/P7 and nested PCR using primer pairs P1/P7-R16F2n/R16R2 and P1/P7-fU5/rU3. The PCR products were sequenced and subsequent analysis using GenBank database information at the national center for biotechnology was employed. Results and Discussion: Comparison of different DNA extraction methods indicated using suitable method can significantly reduce false negative reaction, but even in successful column-based DNA extraction method, false negative reactions were reported that were due to low phytoplasma concentration and irregular distribution within host tissues or could be caused by inhibitor presence in DNA samples. Based on results of sequencing, false positives were obtained sporadically, using primer pairs combination P1/P7- R16F2n/R16R2 that may be arising from cross over contamination or sequence homology with plant genome, so some primers can react probably with sequences of plant genome and false positives could be observed. Since false positives are also a major problem in PCR protocols, especially in nested PCR so single tube nested PCR (STNP) was optimized to avoid false positive reaction but regardless advantages of this method such as facility, cost and time effective and ability to detect low concentration of pathogen, false positive reactions were observed in a few samples. The advantage of STNP is that tubes do not have to be opened, so the risk of contamination minimized. Conclusion: PCR-based techniques for phytoplasma detection, appears to be the method of choice because of their high sensitivity and specificity. Using suitable method for extraction of DNA from infected plant tissues are getting more critical for precise detection through increasing DNA quality and quantity. In the other hand confirmation of phytoplasma presence must be accomplished at least by RFLP analyses or different primer pair combination to avoid false positive detection.