Claudio Ranieri's side looked to be returning to the summit after Francesco Totti's 14th-minute goal but a second-half double from Giampaolo Pazzini sent Samp back up to the fourth and final Champions League qualifying spot.
Inter, who had let slip a nine-point lead from February, beat Atalanta 3-1 on Saturday and lie two points ahead of Roma with three games left as they seek a fifth straight scudetto.
NEWS:Inter second string see off Atalanta
The visit of Samp had looked the toughest-remaining hurdle in debt-laden Roma's fairytale route to a surprise fourth Serie A title and so it proved.
Totti, substituted in last weekend's derby win over Lazio because he was over-excited, expertly turned in Mirko Vucinic's pass to boost calls for the Italian to come out of international retirement at June's World Cup.
But it was another Azzurri striker hoping to be in South Africa who produced stunned silence in the Stadio Olimpico.
First Pazzini headed in former Roma forward Antonio Cassano's cross early in the second half before poking in the winner five minutes from time.
"First half that wasn't us," Pazzini told Sky TV. "Then we sorted it out in the second half. It was a great match and our Champions League dream continues."
Roma, who also face Inter in the Italian Cup final on May 5, probably did enough to claim a draw, with Totti hitting the post and a strong penalty appeal being turned down.
Sampdoria moved two points above Palermo, who beat third-placed AC Milan 3-1 on Saturday, and six above Juventus.
BENITEZ LINKED
Sixth-placed Juve, again linked in the media with Liverpool coach Rafael Benitez for next season, boosted their faint hopes of fourth when Italy striker Vincenzo Iaquinta netted his first goals since September in a 3-0 home win over Bari.
Iaquinta, likely to be in Italy's World Cup squad and possibly the starting lineup, has struggled with injury for most of the season but looked fresh after coming on as a substitute.
He rounded the goalkeeper and scored from a tight angle early in the second half for the opener and prodded in a third.
In between his goals, strike partner Alessandro Del Piero converted a penalty after a foul on playmaker Diego, who had one of his best games for Juve in a difficult first season.
Interim coach Alberto Zaccheroni has failed to win over fans since replacing Ciro Ferrara in January and Juve officials have refused to confirm or deny links with Benitez, whose Liverpool side are enduring just as bad a season as the Turin club.
Bottom side Livorno were relegated despite a 3-1 home victory over Catania and Bologna's 2-1 win over Parma also meant Siena are all but down following a 4-1 defeat at Udinese.
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