(GTG)5 MSP-PCR Fingerprinting as a Technique for Discrimination of Wine Associated Yeasts?

Abstract

In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.

Identification by PCR and evaluation of probiotic potential in yeast strains found in kefir samples in the city of Santa Maria, RS, Brazil

“…The yeasts genomic DNA was extracted according to the methodology proposed by Ramírez-Castrillón et al (2014). The quality of the extracted DNA was analysed on GelRed  stained 1% (w / v) agarose gels and visualized under UV light with the Syngene photodocumentation system.…”

“…The amplification conditions were: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 15 sec, 55 °C for 45 sec, extension at 72 °C for 90 sec, and final extension at 72 °C for 6 min (Mattanna et al, 2014;Ramírez-Castrillón et al, 2014).…”

Bacterial species from retailed poultry eggs in Tshwane, South Africa: Implication for consumers

“…All the strains that are grouped in the same cluster of the sequenced one are assumed to belong to the same species. 26 Salmonella Typhimurium isolates were grouped into two separate clusters. The first cluster consists of Salmonella Typhimurium isolates from samples with code numbers AJ01-AJ06, AJ17-AJ18, AJ22-AJ31, AJ41, AJ43 and AJ44, which were grouped together as one cluster because of the similarity in their band patterns.…”

“…All the strains that have been grouped in the same cluster are assumed to belong to the same species ( Figure 3). 26 Three different serotypes of Salmonella were identified from 47 bacterial isolates that were previously identified by MALDI-TOF ( Figure 3). Salmonella ser.…”

Evaluation of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for differentiation of Pichia kluyveri strains isolated from traditional fermentation processes

“…The fingerprinting method using the primers (GTG) 5 was optimized from Ramírez‐Castrillón et al, using strain Fermichamp ( S. cerevisiae ) as reference strain. The optimized reaction mix for a volume of 10 μL was: 5 μL of JumpStart Taq ReadyMix (Sigma‐Aldrich), 0.8 μL (10 mM) primer, 3.2 μL water and 1 μL of DNA.…”