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Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

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(b) IS900 Nested PCR, using p90-p91 primers 4 for <str<strong>on</strong>g>the</str<strong>on</strong>g> first round followed by a sec<strong>on</strong>d round<br />

with specific primers (see Table 1). The first round <str<strong>on</strong>g>of</str<strong>on</strong>g> Nested PCR was performed in 25 μl<br />

final volume with 0.1 U <str<strong>on</strong>g>of</str<strong>on</strong>g> TAQ (Qiagen, Milan, Italy) adding two μl <str<strong>on</strong>g>of</str<strong>on</strong>g> DNA. The sec<strong>on</strong>d<br />

round was performed by adding two μl <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> previous run in a final volume <str<strong>on</strong>g>of</str<strong>on</strong>g> 25 μl. The<br />

reacti<strong>on</strong> was carried out in Mastercycler ep gradient s (Eppendorf);<br />

(c) IS900 TaqMan Real time PCR, performed adding two μl <str<strong>on</strong>g>of</str<strong>on</strong>g> DNA in 25 μl final volume,<br />

using TaqMan® Gene Expressi<strong>on</strong> Master Mix (Applied Biosystems, Milan, Italy) in<br />

StepOnePlus Real-Time PCR System (Applied Biosystems). Fluorescence intensity<br />

was normalized with ROX dye;<br />

(d) f57 SYBR Green Real Time PCR, performed adding two μl <str<strong>on</strong>g>of</str<strong>on</strong>g> DNA in 25 μl final volume<br />

using Power SYBR® Green PCR Master Mix (Applied Biosystems) in StepOnePlus<br />

Real-Time PCR System (Applied Biosystems). Fluorescence intensity was normalized<br />

with ROX dye. The amplificati<strong>on</strong> was followed by melting curve analysis, melting <str<strong>on</strong>g>the</str<strong>on</strong>g><br />

amplic<strong>on</strong> from 60 °C to 95 °C and recording <str<strong>on</strong>g>the</str<strong>on</strong>g> fluorescence 3.3 times per degree with a<br />

temperature ramp rate <str<strong>on</strong>g>of</str<strong>on</strong>g> 2.2 °C/sec.<br />

Both <str<strong>on</strong>g>the</str<strong>on</strong>g> commercial PCR (a) and <str<strong>on</strong>g>the</str<strong>on</strong>g> TaqMan (c) c<strong>on</strong>tained an internal amplificati<strong>on</strong> c<strong>on</strong>trol<br />

in order to discriminate between negative or inhibited samples, while <str<strong>on</strong>g>the</str<strong>on</strong>g> o<str<strong>on</strong>g>the</str<strong>on</strong>g>r test did not<br />

c<strong>on</strong>tain any internal amplificati<strong>on</strong> c<strong>on</strong>trol. Primers sequences are listed in table 1.<br />

Table 1: Primers and probes sequences.<br />

Methods Oligo name Sequences Ta<br />

(°C)<br />

a Adiavet Kit primers unknown 62<br />

b<br />

P90 5’-GAAGGGTGTTCGGGGCCGTCGCTTAGG-3’<br />

P91 5’-GGCGTTGAGGTCGATCGCCCACGTGAT-3’<br />

Nested Forward 5'-GACGACTCGACCGCTAATTG-3'<br />

Nested Reverse 5'-CCGTAACCGTCATTGTCCAG-3'<br />

62<br />

58<br />

IS900 Forward 5’-CCGGTAAGGCCGACCATTA-3’<br />

c IS900 Reverse 5’-ACCCGCTGCGAGAGCA-3’<br />

60<br />

IS 900 Probe 6-FAM-CATGGTTATTAACGACGACGCGCAGC-TAMRA-3’<br />

d<br />

f57 Forward<br />

f57 Reverse<br />

5'-ATAGCTTTCCTCTCCTTCGTC-3'<br />

5'-CAGGGCAACAACATATTCGG-3'<br />

60<br />

RESULTS<br />

Out <str<strong>on</strong>g>of</str<strong>on</strong>g> 37 milk samples tested we found <strong>on</strong>ly <strong>on</strong>e positive sample (3%) using (a), (c) and (d)<br />

methods. The Nested PCR method (b) showed a total <str<strong>on</strong>g>of</str<strong>on</strong>g> eight positive samples (22%). No<br />

inhibited samples were found with (a) and (c) methods.<br />

(a) Adiavet PCR. Note <str<strong>on</strong>g>the</str<strong>on</strong>g> internal amplificati<strong>on</strong> c<strong>on</strong>trol in all samples. Only<br />

<strong>on</strong>e sample (4) was positive. M: Marker; NE: negative extracti<strong>on</strong> c<strong>on</strong>trol; NN:<br />

no template c<strong>on</strong>trol; 1-9: milk samples.<br />

M NE 1 2 3 4 5 6 7 8 9 NN<br />

57

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