In Progress / Literature-based Diagnostics:
Molecular: A "universal" PCR assay has been developed that enables amplification of the 16S rRNA genes of phytoplasmas. Digestion of these PCR products with selected restriction enzymes provides a DNA fingerprint in the form of 16S rDNA fragment patterns that can be used to determine phytoplasma identity (Ahrens and Seemuller, 1992; Deng and Hiruki, 1991; Lee et al., 1993; Lee et al., 1995; Schneider et al., 1995; Gundersen and Lee, 1996; Smart et al., 1996; Gibb et al., 1999; Jarausch et al., 2000; Heinrich et al., 2001).
Seemuller and Schneider (2004) offer a summary of the molecular studies conducted on on the apple proliferation, European stone fruit yellows, and pear decline phytoplasmas.
Nested and Species-specific PCR: Torres et al. (2004) used nested PCR with 16SrX group specific primers and were able to detect the ESFY phytoplasma in asymptomatic trees.
A primer pair (ECA1/ ECA 2), designed from conserved chromosomal sequences, showed no cross reaction in PCR amplification with other phytoplasmas of the apple proliferation group and proved to be highly specific for ESFY phytoplasma (Jarausch et al., 1998).
Rubio-Cabetas and Sancho (2009) evaluated nested PCR with group-specific primers followed by RFLP and direct PCR with specific primers for Ca. Phytoplasma prunorum from Jarausch et al. (1998). Rubio-Cabetas and Sancho (2009) recommended the nested PCR followed by RFLP analysis for routine diagnosis rather than the direct PCR.
PCR-ELISA: Poggi Pollini et al. (1997) used immunoenzymatic detection of PCR products to detect phytoplasmas, including ESFY.
Co-operational PCR: Bertolini et al. (2007) developed a co-operational PCR coupled with dot blot hybridization for detection of Ca. Phytoplasma mali, Ca. Phytoplasma prunorum, and Ca. Phytoplasma pyri. The sensitivity of this method was at least one hundred times greater than conventional PCR and similar to that achieved by nested PCR and real-time PCR.
Real-time PCR: Torres et al. (2005) developed a real-time PCR that detects Ca. Phytoplasma mali, Ca. Phytoplasma prunorum, and Ca.Phytoplasma pyri (three pytoplasmas in apple proliferation group of quarantine importance). Martini et al. (2007) and Yvon et al. (2009) developed a specific PCR and real-time PCR assay for Ca. Phytoplasma prunorum in plants and insect vectors. Pignatta et al. (2008) developed a specific multiplex real-time PCR procedure that allows the simultaneous detection of ESFY phytoplasma and host DNA, in order to avoid false negatives due to PCR inhibition.