EPPO-Q-bank

The barcoding protocol within Xylella

Conventional PCR on mutS for Xylella fastidiosa. 

The test is based on the PCR amplification and sequencing of 815 bp (amplicon including primers, determined in in silico analysis) of the DNA mismatch repair protein (mutS) gene and can be used for the identification of Xylella fastidiosa and its subspecies.
  The mutS gene is located on the chromosome. 

Primer sequences and their application are given in the table below.

*Lower-case characters indicate the universal M13 tails. These tails play no role in amplification of the target but are used for generating cycle sequence products.

Master mixes are prepared according to the table below. 

* The MyFi^TM mastermix is supplied as a 2x formulation containing MyFi DNA Polymerase, dNTPs, MgCl₂ and enhancers at optimal concentrations. Other verified PCR master mixes containing a polymerase with proof reading activity are also fit for purpose. 

PCR profile: 2 min at 98°C then 30x [10 s at 98°C, 15 s at 60°C, 30 s at 72°C], 5 min at 72°C, hold at 4°C.

Cycle sequencing reactions are performed using the primers targeting the respective M13 tags in separate reactions. 

The mutS gene is a protein-coding region. Translation Table 11 (Bacterial, Archaeal and Plant Plastid Code) applies to the bacterial mutS gene. 

The mutS amplicon extends from position 1793 to position 2607 included on the positive strand of the gene (2607 bp). 

The mutS barcode (772 nt) extends from position 1813 to position 2584 included on the positive strand of the gene. The mutS barcode starts with AATGAC(A/C)T(C/T) and ends with AAGCGCT(C/T)T. 

See appendix 7 of PM7/129 for guidance on data-analysis.

Performance characteristics

Analytical sensitivity 

Cell pellets of pure cultures are used for the DNA extraction.

DNA concentration of approximately 1,1 ng/µL is sufficient to generate an amplicon that can be sequenced, leading to a consensus sequence with a HQ base call accuracy (Phred > 40) of at least 84%.

Analytical specificity

The analytical specificity of the mutS PCR was in silico assessed on 77 strains of Xylella fastidiosa,  

The mutS PCR is exclusive for Xylella fastidiosa. The only other species in the genus, Xylella taiwanensis, will not produce the amplicon.

The mutS DNA barcode can be used to identify the subspecies of Xylella fastidiosa. 

* These strains cannot be unambiguously assigned to a subspecies on the basis of whole genome sequence

Selectivity

Selectivity does not apply as pure cultures are used.

Reproducibility data

The DNA samples were processed by different persons. Therefore in this situation the reproducibility is identical to diagnostic sensitivity.