4 MATRIZ DE DOCUMENTACION Y DATOS (MDD) - Metodología de Reportes Gerenciales

Federica Fois – “Prevalence of Salmonella spp and Yersinia enterocolitica in slaughtered pigs: molecular typing, virulence profile and antimicrobial resistance” Tesi di Dottorato in “Produzione,

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PCR was performed in 25 µl volumes consisting of 1 µl of DNA template, 0.2 mM

concentrations of deoxynucleoside triphosphates (Sigma Aldrich), 5 X Green Go

Taq® Flexi buffer (Promega), 3 mM MgCl2 (Invitrogen), 1.25 U/µl Go Taq® Hot Start

polymerase (Promega), 1 µM concentrations of each forward and reverse ystA and

ystB (Sigma Aldrich), 2 µM concentrations of each forward and reverse primer inv

(Sigma Aldrich). Primers used for detection of virulence genes are shown in Table 4.

The thermal cycling conditions were the following: 1 cycle of denaturation at 95°C

for 10 min, 25 cycles of melting at 95°C for 15 s, annealing at 62°C for 30 s and

elongation at 72°C for 30 s; final extension at 72°C for 10 min. Amplification was

performed in a GeneAmp PCR System 9700 (Applied Biosystems, USA). For

amplification reaction, a negative control containing water was run in parallel.

Moreover, Y. enterocolitica ATCC 23715 was used as positive control. After

amplification, 10 µl of amplified products were analyzed by electrophoresis on a

2% agarose gel in 1X TAE Electrophoresis buffer at 4V/cm for 2 h. Gel images were

acquired using a Gel Doc digital photo-documentation system (Bio Rad Lab.,

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Y

ERSINIA ENTEROCOLITICA

P

ULSED

F

IELD

G

E

LECTROPHORESIS

(PFGE)

As previously said, PFGE was performed in the laboratories of FoodBorne Infections,

Department of Microbiology and Infection Control, Statens Serum Institut,

Copenhagen (Denmark). All Y. enterocolitica strains were streaked onto SSI to evaluate

purity and incubated at 37°C for 24 h. On this indicator medium, typical Y.

enterocolitica colonies appear small, round, convex and pale like “pearls on a string”.

PFGE was performed on a subset of 32 Y. enterocolitica strains isolated from

slaughtered pigs using the internationally standardized protocol Pulse-Net. An isolate

Y. enterocolitica colony was streaked onto 5% blood agar plate (SSI, Copenhagen) and

incubated at 28°C for 14-18 h. Colonies were transferred into 2 ml of Cell Suspension

Buffer (CSB) and the cell concentrations were adjusted using a spectrophotometer

(Sherwood Scientific, Ltd). Agarose plugs were prepared dissolving 1% Seakem Gold

agarose (Lonza, Rockland–ME) in TE Buffer and placed into a water bath (55-60°C) to

equilibrate. Before preparing plugs, 10% SDS (SSI, Copenhagen) pre–heated to 55 C,

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number. 400 µl of melted 1% Seakem Gold agarose were mixed with 400 µl cell

suspension plus Proteinase K (20 mg/ml). Afterward, the mixture was dispensed into

the wells of reusable plug molds and allowed to solidify at room temperature for 10-15

minutes. For the lysis of cells in agarose plugs, 5 ml of Cell Lysis Buffer were dispensed

into a 50 ml polypropylene screw-cap and Proteinase K (20 mg/ml) was added. Plugs

were transferred from moulds to tube containing Cell Lysis Buffer and incubated in a

shaker water bath at 54–55°C for 2 h. After the incubation period, Cell Lysis Buffer was

removed, sterile Ultrapure Water (CLRW) was added to each tube and the tubes were

then incubated in a shaker water bath at 54–55°C for 20 minutes; wash step with pre-

heated water was performed twice. Afterwards, water was removed and plugs were

washed four times with sterile TE Buffer and incubated in a shaker water bath at 54–

55°C for 20 minutes every time. When the washing step was completed, plugs were

stored until use in TE Buffer at 4°C. For restriction digestion of DNA, plugs were placed

on a large glass slide and from each test sample a slice was cut and placed into 1.5 ml

microcentrifuge tube. Then, 200 µl of restriction enzyme master mix with 50 UI per

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The agarose gel was prepared by dissolving 1% Seakem Gold agarose (Lonza,

Rockland–ME) in TBE Buffer and placed into a water bath (55-60°C) to equilibrate.

Restricted plug slices were removed from 37°C incubator, removed from tubes, loaded

on the bottom of the comb teeth and sealed to the comb with 1% Seakem Gold

agarose (Lonza, Rockland–ME). Then the comb was positioned in the gel form, the gel

was poured and allowed to solidify for 30–45 minutes. Salmonella ser. Braenderup

H9812 was used as molecular weight standard. Freshly prepared TBE was added to the

electrophoresis chamber and chilled to 14°C approximately 30 minutes before the gel

was to be run. Electrophoresis was performed using the following settings: initial

switch time 1.8 s, final switch time 18.7 s, a gradient of 6V and 21 h of electrophoresis.

Gels were stained in ethidium bromide for 20–30 minutes and images were acquired

with GeneSnap software (Syngene, Cambridge, United Kingdom). Comparison of

patterns was performed using BioNumerics software v7.1 (Applied Maths, Sint-

Martens-Platen, Belgium) and cluster analysis was carried out using the Dice similarity

coefficient, with 0.5% optimization and 1.5% tolerance, and the unweighted pair group

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In document Metodología de Reportes Gerenciales (página 37-49)