Otman Bakkal settled PSV's home win over Utrecht nine minutes into the second half when he headed home a perfect cross from Stanislav Manolev to leave the Eindhoven club on 61 points from 28 matches.
Champions Twente, who sit a point behind after the same amount of matches and just six remaining, had a Theo Janssen double to thank as they sealed a 2-0 win.
Janssen opened the scoring nine minutes before the interval and midway through the second half converted a penalty to double the lead after Kaj Ramsteijn was sent off for a last-man foul on Luuk de Jong.
The top two clubs will meet in the next round of fixtures in Enschede on April 2.
Ajax, on 55 points from 28 matches, fell further behind after an 87th minute winner by Timothy Derijck.
Defensive blunders by Gregory van der Wiel and Toby Alderweireld gave Frantisek Kubik the opportunity to put ADO ahead after 31 minutes but central defender Jan Vertonghen equalised 20 minutes before time.
Minutes later Lex Immers restored the lead, and while Christian Eriksen seemed to salvage a point for Ajax five minutes before time, Derijck's strike gave ADO two league wins over Ajax this season for the first time.
Seventh-place Roda JC Kerkrade beat Feyenoord 3-0 for their first home win over the struggling Rotterdam side in 10 years.
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