Have a look at Regex with variables?. From what I understood this might be what you need.
Cheers, CombatSquirrel.
Update: Or maybe in your case, the following might help as well:

#!perl

use strict;
use warnings;

my ($curr, $probe, $target, $pos, $sense);

for (<DATA>) {
   if (/>probe:(\w+):(\w+):\d+:\d+;\s+Interrogation_Position=(\d+);\s+
+(\w+);/) {
      ($probe, $target, $pos, $sense) = ($1, $2, $3, $4);
      if ($curr and $curr ne $target) {
         if ($curr) {
            ### do processing for mismatch here
         }
         $curr = $target;
      } else {
         $curr = $target if (!$curr);
         ### do processing for target here
      }
   } else {
      ### do processing for probe sequence here
   }
}

__DATA__
>probe:MOE430A:1415670_at:549:177; Interrogation_Position=2436; Antise
+nse;
GGCTGATCACATCCAAAAAGTCATG
>probe:MOE430A:1415670_at:549:177; Interrogation_Position=2513; Antise
+nse;
GAGGAAACGTTCACCCTGTCTACTA
>probe:MOE430A:1415670_at:467:433; Interrogation_Position=2521; Antise
+nse;
GTTCACCCTGTCTACTATCAAGACA
>probe:MOE430A:1415670_at:254:643; Interrogation_Position=2533; Antise
+nse;
TACTATCAAGACACTCGAAGAGGCT
>probe:MOE430A:1415670_at:54:269; Interrogation_Position=2556; Antisen
+se;
CTGTGGGCAATATTGTGAAGTTCCT
>probe:MOE430A:1415670_at:405:339; Interrogation_Position=2583; Antise
+nse;
GAATGCATCCTTGTGAGAGGTCAGA
>probe:MOE430A:1415670_at:60:395; Interrogation_Position=2597; Antisen
+se;
GAGAGGTCAGACAAAGTGCCAGAAA
>probe:MOE430A:1415670_at:284:165; Interrogation_Position=2619; Antise
+nse;
AAAACAAGAACACCCACACGCTGCT
>probe:MOE430A:1415670_at:622:145; Interrogation_Position=2634; Antise
+nse;
ACACGCTGCTGCTAGCTGGAGTATT
>probe:MOE430A:1415670_at:291:661; Interrogation_Position=2804; Antise
+nse;
TATCTTGTCCAACACTACGTCGAAG
>probe:MOE430A:1415670_at:146:701; Interrogation_Position=2956; Antise
+nse;
TTGTCACCATGCCTGCAAGGAGAGA
>probe:MOE430A:1415671_at:116:525; Interrogation_Position=1156; Antise
+nse;
GGAACAGGAATGTCGCAACATCGTA
>probe:MOE430A:1415671_at:655:137; Interrogation_Position=1173; Antise
+nse;
ACATCGTATGGATTGCTGAGTGCAT
>probe:MOE430A:1415671_at:398:139; Interrogation_Position=1232; Antise
+nse;
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