Shrewsbury, who had scored 13 goals without reply in their previous ties, could not find a way past their League One opponents after a goalless draw in 90 minutes.
Holt and Ben Davies twice missed good chances and Brighton's young goalkeeper John Sullivan kept the Seagulls in the game with a spectacular flying save to tip over a 20-yard drive from Jamie White.
When the tie went to penalties, the two sides scored their first four. But after Matt Richards scored a fifth for Brighton, Holt, who has scored from the penalty spot six times this season, missed the vital kick.
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