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char = u;code = 117 char = b;code = 98 char = s;code = 115 char = t;code = 116 char = i;code = 105 char = t;code = 116 char = u;code = 117 char = t;code = 116 char = i;code = 105 char = o;code = 111 char = n;code = 110 char = ,;code = 44 char = ;code = 32 char = c;code = 99 char = o;code = 111 char = r;code = 114 char = r;code = 114 char = e;code = 101 char = s;code = 115 char = p;code = 112 char = o;code = 111 char = n;code = 110 char = d;code = 100 char = i;code = 105 char = n;code = 110 char = g;code = 103 char = ;code = 32 char = t;code = 116 char = o;code = 111 char = ;code = 32 char = a;code = 97 char = ;code = 32 char = s;code = 115 char = i;code = 105 char = n;code = 110 char = g;code = 103 char = l;code = 108 char = e;code = 101 char = ;code = 32 char = n;code = 110 char = u;code = 117 char = c;code = 99 char = l;code = 108 char = e;code = 101 char = o;code = 111 char = t;code = 116 char = i;code = 105 char = d;code = 100 char = e;code = 101 char = ;code = 32 char = p;code = 112 char = o;code = 111 char = l;code = 108 char = y;code = 121 char = m;code = 109 char = o;code = 111 char = r;code = 114 char = p;code = 112 char = h;code = 104 char = i;code = 105 char = s;code = 115 char = m;code = 109 char = ;code = 32 char = (;code = 40 char = S;code = 83 char = N;code = 78 char = P;code = 80 char = );code = 41 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = p;code = 112 char = o;code = 111 char = r;code = 114 char = t;code = 116 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = a;code = 97 char = n;code = 110 char = ;code = 32 char = e;code = 101 char = a;code = 97 char = r;code = 114 char = l;code = 108 char = i;code = 105 char = e;code = 101 char = r;code = 114 char = ;code = 32 char = s;code = 115 char = t;code = 116 char = u;code = 117 char = d;code = 100 char = y;code = 121 char = ,;code = 44 char = ;code = 32 char = w;code = 119 char = a;code = 97 char = s;code = 115 char = ;code = 32 char = o;code = 111 char = b;code = 98 char = s;code = 115 char = e;code = 101 char = r;code = 114 char = v;code = 118 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = w;code = 119 char = a;code = 97 char = r;code = 114 char = d;code = 100 char = ;code = 32 char = p;code = 112 char = r;code = 114 char = i;code = 105 char = m;code = 109 char = e;code = 101 char = r;code = 114 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = o;code = 111 char = n;code = 110 char = e;code = 101 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = s;code = 115 char = t;code = 116 char = u;code = 117 char = d;code = 100 char = i;code = 105 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = a;code = 97 char = l;code = 108 char = c;code = 99 char = o;code = 111 char = h;code = 104 char = o;code = 111 char = l;code = 108 char = ;code = 32 char = d;code = 100 char = e;code = 101 char = h;code = 104 char = y;code = 121 char = d;code = 100 char = r;code = 114 char = o;code = 111 char = g;code = 103 char = e;code = 101 char = n;code = 110 char = a;code = 97 char = s;code = 115 char = e;code = 101 char = ;code = 32 char = 1;code = 49 char = ;code = 32 char = (;code = 40 char = A;code = 65 char = d;code = 100 char = h;code = 104 char = 1;code = 49 char = );code = 41 char = ;code = 32 char = (;code = 40 char = 7;code = 55 char = 0;code = 48 char = );code = 41 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = s;code = 115 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = a;code = 97 char = ;code = 32 char = l;code = 108 char = a;code = 97 char = r;code = 114 char = g;code = 103 char = e;code = 101 char = ;code = 32 char = n;code = 110 char = u;code = 117 char = m;code = 109 char = b;code = 98 char = e;code = 101 char = r;code = 114 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = v;code = 118 char = a;code = 97 char = r;code = 114 char = i;code = 105 char = e;code = 101 char = t;code = 116 char = i;code = 105 char = e;code = 101 char = s;code = 115 char = .;code = 46 char = ;code = 32 char = T;code = 84 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = S;code = 83 char = N;code = 78 char = P;code = 80 char = ;code = 32 char = p;code = 112 char = r;code = 114 char = e;code = 101 char = s;code = 115 char = e;code = 101 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = i;code = 105 char = s;code = 115 char = ;code = 32 char = c;code = 99 char = o;code = 111 char = n;code = 110 char = s;code = 115 char = i;code = 105 char = s;code = 115 char = t;code = 116 char = e;code = 101 char = n;code = 110 char = t;code = 116 char = ;code = 32 char = w;code = 119 char = i;code = 105 char = t;code = 116 char = h;code = 104 char = ;code = 32 char = a;code = 97 char = ;code = 32 char = p;code = 112 char = o;code = 111 char = o;code = 111 char = r;code = 114 char = ;code = 32 char = P;code = 80 char = C;code = 67 char = R;code = 82 char = ;code = 32 char = p;code = 112 char = e;code = 101 char = r;code = 114 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = a;code = 97 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = o;code = 111 char = b;code = 98 char = s;code = 115 char = e;code = 101 char = r;code = 114 char = v;code = 118 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = i;code = 105 char = s;code = 115 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ;code = 32 char = a;code = 97 char = l;code = 108 char = o;code = 111 char = n;code = 110 char = g;code = 103 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = e;code = 101 char = s;code = 115 char = t;code = 116 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = v;code = 118 char = a;code = 97 char = r;code = 114 char = i;code = 105 char = e;code = 101 char = t;code = 116 char = i;code = 105 char = e;code = 101 char = s;code = 115 char = .;code = 46 char = ;code = 32 char = T;code = 84 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = o;code = 111 char = b;code = 98 char = t;code = 116 char = a;code = 97 char = i;code = 105 char = n;code = 110 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = d;code = 100 char = a;code = 97 char = t;code = 116 char = a;code = 97 char = ;code = 32 char = s;code = 115 char = h;code = 104 char = o;code = 111 char = w;code = 119 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = a;code = 97 char = t;code = 116 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = A;code = 65 char = d;code = 100 char = h;code = 104 char = 1;code = 49 char = ;code = 32 char = (;code = 40 char = 7;code = 55 char = 0;code = 48 char = );code = 41 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ;code = 32 char = u;code = 117 char = s;code = 115 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = f;code = 102 char = i;code = 105 char = c;code = 99 char = i;code = 105 char = a;code = 97 char = l;code = 108 char = ;code = 32 char = C;code = 67 char = R;code = 82 char = L;code = 76 char = ;code = 32 char = N;code = 78 char = K;code = 75 char = 6;code = 54 char = 0;code = 48 char = 3;code = 51 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ;code = 32 char = i;code = 105 char = s;code = 115 char = ;code = 32 char = u;code = 117 char = n;code = 110 char = r;code = 114 char = e;code = 101 char = l;code = 108 char = i;code = 105 char = a;code = 97 char = b;code = 98 char = l;code = 108 char = e;code = 101 char = .;code = 46 char = ;code = 32 char = B;code = 66 char = a;code = 97 char = s;code = 115 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = o;code = 111 char = n;code = 110 char = ;code = 32 char = o;code = 111 char = u;code = 117 char = r;code = 114 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = s;code = 115 char = u;code = 117 char = l;code = 108 char = t;code = 116 char = s;code = 115 char = ;code = 32 char = f;code = 102 char = r;code = 114 char = o;code = 111 char = m;code = 109 char = ;code = 32 char = b;code = 98 char = o;code = 111 char = t;code = 116 char = h;code = 104 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = n;code = 110 char = u;code = 117 char = c;code = 99 char = l;code = 108 char = e;code = 101 char = o;code = 111 char = t;code = 116 char = i;code = 105 char = d;code = 100 char = e;code = 101 char = ;code = 32 char = s;code = 115 char = t;code = 116 char = a;code = 97 char = b;code = 98 char = i;code = 105 char = l;code = 108 char = i;code = 105 char = t;code = 116 char = y;code = 121 char = ;code = 32 char = s;code = 115 char = t;code = 116 char = u;code = 117 char = d;code = 100 char = y;code = 121 char = ;code = 32 char = a;code = 97 char = n;code = 110 char = d;code = 100 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = P;code = 80 char = C;code = 67 char = R;code = 82 char = ;code = 32 char = p;code = 112 char = e;code = 101 char = r;code = 114 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = a;code = 97 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = e;code = 101 char = s;code = 115 char = t;code = 116 char = ,;code = 44 char = ;code = 32 char = w;code = 119 char = e;code = 101 char = ;code = 32 char = c;code = 99 char = a;code = 97 char = n;code = 110 char = ;code = 32 char = c;code = 99 char = o;code = 111 char = n;code = 110 char = c;code = 99 char = l;code = 108 char = u;code = 117 char = d;code = 100 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = a;code = 97 char = t;code = 116 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = A;code = 65 char = d;code = 100 char = h;code = 104 char = 1;code = 49 char = ;code = 32 char = (;code = 40 char = 1;code = 49 char = 3;code = 51 char = 6;code = 54 char = );code = 41 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = f;code = 102 char = e;code = 101 char = r;code = 114 char = e;code = 101 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ;code = 32 char = (;code = 40 char = T;code = 84 char = 2;code = 50 char = 5;code = 53 char = ;code = 32 char = a;code = 97 char = n;code = 110 char = d;code = 100 char = ;code = 32 char = B;code = 66 char = t;code = 116 char = 1;code = 49 char = 1;code = 49 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = s;code = 115 char = );code = 41 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = ;code = 32 char = w;code = 119 char = e;code = 101 char = l;code = 108 char = l;code = 108 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = e;code = 101 char = s;code = 115 char = t;code = 116 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = h;code = 104 char = i;code = 105 char = g;code = 103 char = h;code = 104 char = ;code = 32 char = m;code = 109 char = o;code = 111 char = b;code = 98 char = i;code = 105 char = l;code = 108 char = i;code = 105 char = t;code = 116 char = y;code = 121 char = ;code = 32 char = g;code = 103 char = r;code = 114 char = o;code = 111 char = u;code = 117 char = p;code = 112 char = ;code = 32 char = p;code = 112 char = r;code = 114 char = o;code = 111 char = t;code = 116 char = e;code = 101 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = g;code = 103 char = e;code = 101 char = n;code = 110 char = e;code = 101 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ,;code = 44 char = ;code = 32 char = w;code = 119 char = h;code = 104 char = i;code = 105 char = c;code = 99 char = h;code = 104 char = ;code = 32 char = a;code = 97 char = l;code = 108 char = s;code = 115 char = o;code = 111 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = ;code = 32 char = p;code = 112 char = a;code = 97 char = r;code = 114 char = t;code = 116 char = s;code = 115 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = C;code = 67 char = R;code = 82 char = L;code = 76 char = ;code = 32 char = m;code = 109 char = e;code = 101 char = t;code = 116 char = h;code = 104 char = o;code = 111 char = d;code = 100 char = s;code = 115 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = ;code = 32 char = q;code = 113 char = u;code = 117 char = a;code = 97 char = n;code = 110 char = t;code = 116 char = i;code = 105 char = f;code = 102 char = i;code = 105 char = c;code = 99 char = a;code = 97 char = t;code = 116 char = i;code = 105 char = o;code = 111 char = n;code = 110 char = ,;code = 44 char = ;code = 32 char = a;code = 97 char = r;code = 114 char = e;code = 101 char = ;code = 32 char = h;code = 104 char = i;code = 105 char = g;code = 103 char = h;code = 104 char = l;code = 108 char = y;code = 121 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = l;code = 108 char = i;code = 105 char = a;code = 97 char = b;code = 98 char = l;code = 108 char = e;code = 101 char = .;code = 46 char = ;code = 32 char = D;code = 68 char = e;code = 101 char = s;code = 115 char = p;code = 112 char = i;code = 105 char = t;code = 116 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = o;code = 111 char = b;code = 98 char = s;code = 115 char = e;code = 101 char = r;code = 114 char = v;code = 118 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = u;code = 117 char = n;code = 110 char = i;code = 105 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = i;code = 105 char = t;code = 116 char = y;code = 121 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = n;code = 110 char = u;code = 117 char = c;code = 99 char = l;code = 108 char = e;code = 101 char = o;code = 111 char = t;code = 116 char = i;code = 105 char = d;code = 100 char = e;code = 101 char = ;code = 32 char = s;code = 115 char = e;code = 101 char = q;code = 113 char = u;code = 117 char = e;code = 101 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = v;code = 118 char = e;code = 101 char = r;code = 114 char = t;code = 116 char = a;code = 97 char = s;code = 115 char = e;code = 101 char = ;code = 32 char = g;code = 103 char = e;code = 101 char = n;code = 110 char = e;code = 101 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ,;code = 44 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = e;code = 101 char = ;code = 32 char = P;code = 80 char = C;code = 67 char = R;code = 82 char = ;code = 32 char = p;code = 112 char = e;code = 101 char = r;code = 114 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = a;code = 97 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = e;code = 101 char = s;code = 115 char = t;code = 116 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = v;code = 118 char = e;code = 101 char = a;code = 97 char = l;code = 108 char = s;code = 115 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = a;code = 97 char = t;code = 116 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = i;code = 105 char = s;code = 115 char = ;code = 32 char = t;code = 116 char = a;code = 97 char = r;code = 114 char = g;code = 103 char = e;code = 101 char = t;code = 116 char = ;code = 32 char = s;code = 115 char = e;code = 101 char = q;code = 113 char = u;code = 117 char = e;code = 101 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = m;code = 109 char = i;code = 105 char = g;code = 103 char = h;code = 104 char = t;code = 116 char = ;code = 32 char = o;code = 111 char = c;code = 99 char = c;code = 99 char = u;code = 117 char = r;code = 114 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = m;code = 109 char = o;code = 111 char = r;code = 114 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = h;code = 104 char = a;code = 97 char = n;code = 110 char = ;code = 32 char = o;code = 111 char = n;code = 110 char = e;code = 101 char = ;code = 32 char = c;code = 99 char = o;code = 111 char = p;code = 112 char = y;code = 121 char = .;code = 46 char = ;code = 32 char = F;code = 70 char = i;code = 105 char = n;code = 110 char = a;code = 97 char = l;code = 108 char = l;code = 108 char = y;code = 121 char = ,;code = 44 char = ;code = 32 char = a;code = 97 char = l;code = 108 char = t;code = 116 char = h;code = 104 char = o;code = 111 char = u;code = 117 char = g;code = 103 char = h;code = 104 char = ;code = 32 char = c;code = 99 char = u;code = 117 char = r;code = 114 char = r;code = 114 char = e;code = 101 char = n;code = 110 char = t;code = 116 char = l;code = 108 char = y;code = 121 char = ;code = 32 char = n;code = 110 char = o;code = 111 char = t;code = 116 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = i;code = 105 char = n;code = 110 char = g;code = 103 char = ;code = 32 char = a;code = 97 char = ;code = 32 char = p;code = 112 char = a;code = 97 char = r;code = 114 char = t;code = 116 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = f;code = 102 char = i;code = 105 char = c;code = 99 char = i;code = 105 char = a;code = 97 char = l;code = 108 char = ;code = 32 char = q;code = 113 char = u;code = 117 char = a;code = 97 char = n;code = 110 char = t;code = 116 char = i;code = 105 char = f;code = 102 char = i;code = 105 char = c;code = 99 char = a;code = 97 char = t;code = 116 char = i;code = 105 char = o;code = 111 char = n;code = 110 char = ;code = 32 char = m;code = 109 char = e;code = 101 char = t;code = 116 char = h;code = 104 char = o;code = 111 char = d;code = 100 char = s;code = 115 char = ,;code = 44 char = ;code = 32 char = z;code = 122 char = e;code = 101 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = a;code = 97 char = n;code = 110 char = d;code = 100 char = ;code = 32 char = S;code = 83 char = S;code = 83 char = I;code = 73 char = I;code = 73 char = b;code = 98 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = s;code = 115 char = ;code = 32 char = a;code = 97 char = r;code = 114 char = e;code = 101 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = u;code = 117 char = n;code = 110 char = d;code = 100 char = ;code = 32 char = t;code = 116 char = o;code = 111 char = ;code = 32 char = b;code = 98 char = e;code = 101 char = ;code = 32 char = h;code = 104 char = i;code = 105 char = g;code = 103 char = h;code = 104 char = l;code = 108 char = y;code = 121 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = l;code = 108 char = i;code = 105 char = a;code = 97 char = b;code = 98 char = l;code = 108 char = e;code = 101 char = ;code = 32 char = i;code = 105 char = n;code = 110 char = ;code = 32 char = t;code = 116 char = e;code = 101 char = r;code = 114 char = m;code = 109 char = s;code = 115 char = ;code = 32 char = o;code = 111 char = f;code = 102 char = ;code = 32 char = n;code = 110 char = u;code = 117 char = c;code = 99 char = l;code = 108 char = e;code = 101 char = o;code = 111 char = t;code = 116 char = i;code = 105 char = d;code = 100 char = e;code = 101 char = ;code = 32 char = s;code = 115 char = t;code = 116 char = a;code = 97 char = b;code = 98 char = i;code = 105 char = l;code = 108 char = i;code = 105 char = t;code = 116 char = y;code = 121 char = ;code = 32 char = a;code = 97 char = n;code = 110 char = d;code = 100 char = ;code = 32 char = P;code = 80 char = C;code = 67 char = R;code = 82 char = ;code = 32 char = p;code = 112 char = e;code = 101 char = r;code = 114 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = m;code = 109 char = a;code = 97 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = a;code = 97 char = n;code = 110 char = d;code = 100 char = ;code = 32 char = a;code = 97 char = r;code = 114 char = e;code = 101 char = ;code = 32 char = p;code = 112 char = r;code = 114 char = o;code = 111 char = p;code = 112 char = o;code = 111 char = s;code = 115 char = e;code = 101 char = d;code = 100 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = ;code = 32 char = g;code = 103 char = o;code = 111 char = o;code = 111 char = d;code = 100 char = ;code = 32 char = a;code = 97 char = l;code = 108 char = t;code = 116 char = e;code = 101 char = r;code = 114 char = n;code = 110 char = a;code = 97 char = t;code = 116 char = i;code = 105 char = v;code = 118 char = e;code = 101 char = ;code = 32 char = t;code = 116 char = a;code = 97 char = r;code = 114 char = g;code = 103 char = e;code = 101 char = t;code = 116 char = s;code = 115 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = ;code = 32 char = a;code = 97 char = ;code = 32 char = r;code = 114 char = e;code = 101 char = f;code = 102 char = e;code = 101 char = r;code = 114 char = e;code = 101 char = n;code = 110 char = c;code = 99 char = e;code = 101 char = ;code = 32 char = a;code = 97 char = s;code = 115 char = s;code = 115 char = a;code = 97 char = y;code = 121 char = ;code = 32 char = f;code = 102 char = o;code = 111 char = r;code = 114 char = ;code = 32 char = m;code = 109 char = a;code = 97 char = i;code = 105 char = z;code = 122 char = e;code = 101 char = .;code = 46 char = <;code = 60 char = b;code = 98 char = r;code = 114 char = /;code = 47 char = >;code = 62 result sql = A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). WE ANALYSED THE NUCLEOTIDE SEQUENCE VARIATION IN THE TARGET GENOMIC REGIONS IN A BROAD RANGE OF TRANSGENIC AND CONVENTIONAL VARIETIES AND LINES: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.
Publication: Anal Bioanal Chem 2010 Mar ;396(6)::2189-201. :doi: 10.1007/s00216-009-3386-4.
Publication Date: 2010 Mar
Study Author(s): Papazova, Nina;Zhang, David;Gruden, Kristina;Vojvoda, Jana;Yang, Litao;Buh Gasparic, Meti;Blejec, Andrej;Fouilloux, Stephane;De Loose, Marc;Taverniers, Isabel;
Institution: Technology and Food Sciences, Institute for Agricultural and Fisheries Research (ILVO), Burg. Van Gansberghelaan 115, 9820 Merelbeke, Belgium.
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A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). WE ANALYSED THE NUCLEOTIDE SEQUENCE VARIATION IN THE TARGET GENOMIC REGIONS IN A BROAD RANGE OF TRANSGENIC AND CONVENTIONAL VARIETIES AND LINES: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.
PMID: 20069284