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Determinazione di correlazioni genetiche mediante tecnica MLVA

Determinazione di correlazioni genetiche mediante tecnica MLVA fra popolazioni di Pseudomonas syringae pv. actinidiae (Psa) con differente origine geografica

Ente finanziatore. Risorse dei ricercatori coinvolti (University of Otago , NZ – Unitus DAFNE)
Unità di Ricerca DAFNE UNITUS
Responsabile scientifico Prof. GM BALESTRA
Partecipanti: Dr. A. Mazzaglia
Durata 2017 – 2019

At present:

  • we have tested PCR amplification for all the 24 VNTR loci on a pool of 10 strains. We are experiencing problem (amplification failure) with a couple of them, among those from the new paper of Cunty et al. We will try to fix the problem in the next days.
  •  Among those operating regularly, we have amplified 14 VNTR loci on 24, on the first 66 strains from our collection.

1) Visualization of amplified fragment by capillary electrophoresis (Qiaxcel)
We are able to asses the exact dimension of the amplicons (with a 2-3 bp margin of error) by this specifically devoted instrument, producing detailed report for each of them.

At present: all the VNTRs successfully amplified (924 amplicons) were already run.

2) Calculation of the number of TR in each amplicon
To perform the final analysis (MLVA) it is necessary to assess the exact number of specific repeats within the amplicon by subtraction of the flanking region.

At present: all the VNTRs successfully amplified (924 amplicons) were already analysed and the number of TRs calculated.

3) Comparison of TRs number by High Resolution Melting-Real Time PCR analysis (HRM-RT-PCR)
This methodology is able to detect differences up to a single mutation (SNP) within a single amplicon. We plan to use this technique in any doubt case, before moving on sequencing.

At present: not used yet, due to confident results.

7) Confirmation of TR number by sequencing
Anyway, at least 10 strains per each VNTR locus are to be sequenced, in order to confirm the exactness of previous calculations. It is necessary to check for possible differences within flanking
regions that could affect results.

At present: about 70 sequencing was already carried out and are under post-processing refining.

Also, sequencing will be performed each time the results from capillary electrophoresis and HRMRT-PCR pose doubts in interpretation of results.


8) Data assembly and elaboration
Once results will be obtained, data will be processed according to the conventional standards for this methodology.

At present: in MLVA partial analysis, i.e. only few strains or few VNTR loci, doesn’t make much sense, so it will not be performed before complete data acquisition.

Basically data will be presented in form of Minimum Spanning Tree (MST) and/or genetic similarity
dendrograms UPGMA. Statistical analysis (K-means analysis, Discriminant Analysis of Principal
Components DAPC, etc.) will be carried out in support of the outcome.