Tom,
The classic test for queenlessness is challenging a supposedly queenless
colony with the means to make a queen, i.e. very young larvae.  If they
haven't started queen cells within 24 hours they have a queen.  Although it is
frequently and truely stated that bees do nothing invariably this test is more
reliable than most operations that depend on the behavior of bees.
 
Your idea of separating the two brood boxes with queen excluders seems sound
but why not take it a stage further and add a super between as well?  If there
are two queens you can run the hive as a 2 queen system for a while until you
decide which queen is best.  Put the queen you are certain of in the bottom
box. If there is only the one queen the bees may treat the upper brood box as
a super as the brood emerges.  If you don't want to extract from combs that
have been bred in, use the combs of stores for nuclei or for winter stores.
 
Most of my queen excluders have a small slot cut in the rim to act as a drone
escape or an upper entrance.  If not wanted you can always close it with a
wooden wedge or a lump of mud.
Regards,  Chris