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London Irish ended their run of 10 successive defeats in all competitions to gain revenge over the Ospreys for their 27-16 defeat in Round 2.
The Irish were far too physical at the breakdown and far more efficient around the field as they forced the Welsh region into mistake after mistake as they saw their chances of reaching a fourth successive quarter-final go up in smoke.
The Ospreys almost scored in the first minute when Mike Phillips drove for the line only to be held up. That piece of defensive wizardry was the fore-runner of many more over the next 79 minutes.
With skipper Seilala Mapusua leading by example, the ferocity of the home defence shook the Ospreys and made them play largely on the back foot throughout the game.
Dan Bowden and Dan Biggar swapped early penalties before a superb midfield break by England fullback Delon Armitage paved the way for the first Irish try by left wing Sailosi Tagicakibau.
Bowden couldn't convert, but he kicked two more penalties to Biggar's one to give the home side a well deserved 14-6 interval lead. The Ospreys may have thought they were still in touch, but it only took a minute of the second half for their task to get even greater.
Irish worked a midfield move off a line-out win on the Ospreys 22 and Topsy Ojo was brought bursting through the middle to score a wonderful training ground move try. This time Bowden added the conversion and the gap was suddenly 15 points.
Biggar then hit the mark from 51 metres to get the visitors moving again, but then the outside half missed with two much easier shots at goal and finally gave way to Sonny Parker after 55 minutes.
As the game loosened up the Ospreys enjoyed more possession, but still couldn't find a way through the water-tight home defence. James Hook took over the kicking duties and landed a penalty to cut the gap to nine points with 14 minutes left to play, but that was it for the Welsh side.
Even though they played the final nine minutes with hooker James Buckland in the sin-bin, Irish held on for a famous victory that had a sting in the tail through a Ryan Lamb penalty to drive the final nail into the Ospreys' Heineken Cup coffin this season.
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