Table 2.

Most commonly used PCR-based techniques for X. fastidiosa identification.

Molecular method	Advantages	Disadvantages	References
Classic PCR	High sensitivity, specificity and accurate results for the detection of Xylella and its subspecies also in non-axenic conditions. Many applications in molecular analysis. Easy diagnostic interpretation	Unable to quantify the target DNA, only qualitative test. Some metabolites or contaminants in the sample can interfere with PCR performance. PCR conditions must be optimized in each host and environment for better performance	Firrao and Bazzi, 1994; Minsavage et al., 1994; Pooler and Hartung, 1995a; Banks et al., 1999; Rodrigues et al., 2003; Huang and Sherald, 2004; Chen et al., 2005; Travensolo et al., 2005; Hernandez-Martinez et al., 2006; Martinati et al., 2007; Morano et al., 2008; Huang, 2009; Livingston et al., 2010; Melanson et al., 2012; Guan et al., 2015; Bleve et al., 2016
RAPD	Useful to study unknown species where there is not apriori. knowledge of sequencing. Quite useful to detect high polymorphisms. Limited cost, Simple and rapid. Small amount of template DNA required. The amplification products can be further characterized	Markers are dominant. Reproducibility can be low among labs and with different polymerases and facilities, especially when not using random primers with high annealing temperature. In many cases standardization of the protocol for each lab is required. Nowadays this technique is considered obsolete	Denny et al., 1988; Hartung and Civerolo, 1989; Grajalmartin et al., 1993; Chen et al., 1995, 2002; Pooler and Hartung, 1995a; Albibi et al., 1998; Rosato et al., 1998; Banks et al., 1999; Hendson et al., 2001; Lacava et al., 2001; Qin et al., 2001; Su et al., 2008
RFLP	It was one of the first methods used for genetic fingerprinting, The basic RFLP analysis is no longer used. Variations exist such as terminal restriction fragment length polymorphism (TRFLP), which may still have applications related to the characterization of bacteria. By PCR-RFLP, the hybridization step can be skipped	Obsolete technique. Relatively large amount of DNA is required. RFLP approach is tedious and requires numerous steps that may take weeks to yield results. Relatively high cost and low polymorphism	Chen et al., 1992; Rosato et al., 1998; Mehta et al., 2001; Qin et al., 2001; Picchi et al., 2006
qRT-PCR	It allows not only the identification, but also the quantification of bacteria in real time. High sensitivity, specificity and reproducibility. Relatively fast method. It is possible to use also variations in melting temperature to differentiate strains of bacteria	Expensive equipment and reagents are required. Setting up and optimization of the protocol require specific technical skills as well the interpretation of results	Oliveira et al., 2002; Schaad et al., 2002; Bextine et al., 2005; Francis et al., 2006; Bextine and Child, 2007; Choi et al., 2010; Harper et al., 2010; Brady et al., 2012; Guan et al., 2013; Li et al., 2013; Ionescu et al., 2016
SSR	Simple lab procedure, relatively low costs to start, based on PCR termocycler. High level of polymorphism and relatively low amount of target DNA required. Co-dominant markers. The reproducibility is quite good	Previous knowledge of the genomic sequence is required to design specific primers, thus SSRs are limited primarily to economically important species. Point mutations at the site of primer annealing could lead to occurrence of null alleles	Della Coletta-Filho et al., 2001; Coletta and Machado, 2003; Lin et al., 2005, 2013, 2015; Montero-Astua et al., 2007; Montes-Borrego et al., 2015; Francisco et al., 2017
MLST	Highly discriminatory nucleotide sequence based method of characterization based on the sequencing of approximately 450-bp internal fragments of seven housekeeping genes amplified by PCR. This approach is particularly helpful for the typing of bacterial pathogens. The system is very sensitive to discriminate X. fastidiosa subspecies and strains in rapid real time reactions. The major advantage of MLST is the possibility to compare the results obtained in different studies. It may also be used to address basic questions about evolutionary and population biology of bacterial spp.	The analysis of only seven loci may limit the sensitivity, especially when close strains are analyzed. Sequencing of the PCR products using an automated sequencer is required. For that, MLST is not always suitable for routine infection controls or outbreak investigation due to relatively high cost and lack of broad access to high-throughput DNA sequencing	Scally et al., 2005; Schuenzel et al., 2005; Almeida et al., 2008; Yuan et al., 2010; Brady et al., 2012; Nunney et al., 2012, 2013, 2014a,b,c; Parker et al., 2012; Elbeaino et al., 2014; Harris and Balci, 2015; Marcelletti and Scortichini, 2016a; Bergsma-Vlami et al., 2017; Coletta-Filho et al., 2017; Denancé et al., 2017; Kandel et al., 2017
Multiplex PCR	Costs are reduced when compared to standard PCR as well as reaction volumes. It allows rapid detection also of multiple strains simultaneously. Close tube system limits the risk of contamination	Primer design is the critical point, they can interfere each other giving false negative (genes or bacteria undetected). Skilled personnel is required to perform the test	Rodrigues et al., 2003; Choi et al., 2010; Myers et al., 2010; Lopes et al., 2014; Jacques et al., 2016
Nested PCR	Improved sensitivity and specificity when compared with classical PCR methodology. Useful technique for studying molecular epidemiology in the field	The protocol may be a little more difficult to optimize than for standard PCR. More time consuming and expensive than normal PCR. Unable to quantify the target DNA	Pooler et al., 1997; Buzkan et al., 2003; Ciapina et al., 2004; Huang, 2007; Silva et al., 2007; Lopes et al., 2014