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��. ��:

19-Sep-2000

�� , 16(4), ��-�� 1999, 356-367

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�� *

�. ��,¹ �. ��,¹ �. ��,¹
�. ��,¹ �. ��,² �. ��¹

¹�� -��, �� , ��
²�� , ��

�� . � �� , �� Crohn.
Y��-�� -�� (probe), �� DNA ��, �� Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium intracellulare �� Mycobacterium paratuberculosis.
�� , �� , �� . � ��, ��, �� Crohn �� , ��, �� Mycobacterium paratuberculosis �� Mycobacterium tuberculosis complex, �� .
�� . � �� , �� , �� Crohn.

�� : �� Crohn, ��.

� �� , ��' �,�� , �� .^1,2 �� 100 �� Koch, � �� . �� Mycobacterium tuberculosis, �� 90.000.000 �� 1990� 1999,³�� 30.000.000 �� .⁴ �� , � ��, �� , �� , �� .⁵ �� , �� , �� ^6,7 �� Crohn.⁸

� �� , �� , �� . � �� 100 ��/mL ��, �� 3�7 ��.⁹ �� 2�3 ��, �� .¹⁰ �� BACTEC, �� (15�22 ��),¹¹ �� , �� , ��.⁷ � �� Ziehl-Nielsen �� 10.000 ��/mL ��¹² �� 22�78% �� ,⁷ �� Mycobacterium tuberculosis �� .¹³ �� -�� (Gene Probe, San Diego, California, SNAP system, Syngene Inc, San Diego, California), � �� Ziehl-Nielsen �� , � �� .¹⁴

�� , �� (polymerase chain reaction, PCR) �� Mycobacterium tuberculosis complex (M. tuberculosis, �. bovis, M. bovis BCG, M. africanum, M. microti), Mycobacterium avium complex (M. avium MAIS2, M. avium serovar 7, M. avium WP M21, M. paratuberculosis Linda, M. paratuberculosis C286, M. paratuberculosis ATCC 19698) �� Mycobacterium paratuberculosis, �� . � �� , �� , �� Crohn.

�� 

��

�� , �� , �� DNA �� . �� Shropshire (Shropshire Veterinary Investigation Center) �� , �� DNA (��. 1).

�� , � �� (��), �� , �� Crohn. �� :

�� : �� , �� . �� Ziehl-Nielsen, �� . �� 35 �� Ziehl-Nielsen, �� 39 �� .

�� : �� , �� . �� Ziehl-�ielsen, �� . �� Ziehl-Nielsen, �� . �� , �� , �� .

�� : �� , �� Crohn. �� Ziehl-�ielsen, �� . �� , ��.

�� : �� , �� . �� (30), �� (10), �� (4) �� (6). �� /�� . �� Ziehl-Nielsen, �� , �� 22, �� 30 �� , �� .

��

�� DNA. �� 2 mL �� 2�3 �� 10 �m. �� 60 ^oC, �� 75%, �� . �� , �� , �� SDS �� 50 ^oC �� 30 min �� 12 ��.¹⁶ �� -��-�� . �� DNA �� 50 �L �� .¹⁶

�� . �� Mycobacterium tuberculosis complex �� Mycobacterium avium complex �� M. paratuberculosis �� 7H9 Broth �� Middlebrook ADC Enrichment Bacto, �� 2 mL �� . �� , �� 600 nm �� colony forming units (cfu)/mL �� Miles-Misra.^15,17 ��, �� DNA,¹⁸ �� . �� ¹⁹�� DNA/�� , �� .

�� . �� , �� 10 �� Ziehl-Nielsen. � �� , �� (Rhodococcus, Corynebacterium, Nocardia, Actinomyces, E. coli). �� , �� , �� , �� .

�� . �� -��, �� (��. 2), �� , �� (Genbank). �� , �� , �� (NCBI BLAST Search). ��, �� , �� , �� (OLIGO 5).

Mtb1 �� 2: �� 243 �� (��), �� MPB64, �� ²⁰ �� Mycobacterium tuberculosis complex.

Mtb1(184�197): 5�-GCT CTG TTG TTC GGG TGT GGC CA-3�

MTb2 (404�427): 5�-GAT ATT CAA TTC GTA GGG GGC TT-3�

MAC1 �� 2: �� 91 ��, �� IS1110,²¹ �� ²² �� M. avium complex �� M. intracellulare.

MAC1 (1353�1370): 5�-CAG GCA GAG GGT GGC CG-3�

MAC2 (1427�1444): 5�-CGA ATC ACC CCC GAT CA-3�

�16SD2 �� M16SU: �� 509 ��, �� 16S rRNA �� M. intracellulare, �� M. intracellulare, M. avium, M. paratuberculosis, �. smegmatis �� M. gordonae.

�16SD2 (68�93): 5�-GTG GGC AAT CTG CCC TGC ACT TCG G-3�

M16SU (481�668): 5�-GCC CGC ACG CTC ACA GTT AAG CCG T-3�

� �� PCR �� , �� hot start,¹⁶ �� Perkin Elmer 9600. �� , �� 50 �L, �� 50 mM KCl, 10 mM Tris-HCl (pH 8,4 �� ), 1,5 mM MgCl₂, 0,25 �� -��, 200 �� (ATP, TTP, CTP, GTP), 2,5 units Taq ��, 0,05�0,1 �g DNA, �� (HPLC) � �� . �� , � �� (mineral oil). �� , �� 94 ^oC �� 10 min, �� 62 ^oC �� 0,5 min, �� 72 ^oC �� 1 min ��, ��, �� 94 ^oC �� 0,5 min. � �� 30 �� , �� 72 ^oC �� 7 min.

�� (�� , carry over effect²³), � �� -N-�� [UDG (GIBCOBRL)].

�� PCR �� 2%, �� . �� DNA �� film Polaroid �� (��. 1).

�� , �� (20% �� ), �� DNA �� (�� ). �� , �� DNA (�� ). �� , �� , �� . �� (internal control), �� . ��, �� -�� ,²⁴ � �� .

�� , �� . ��, �� , �� .

Southern blotting

�� PCR, �� -�� DNA. � �� , �� , �� ECL (ECL detection system Amersham International plc, Amersham Place, Bucknghamshire, England).

�� , �� DNA �� , �� , � �� (�16SD2 �� M16SU). � �� M. paratuberculosis �� (M. intracellulare, M. avium, M. paratuberculosis, �. smegmatis �� M. gordonae) �� .

PRAVMPAR (132�161): 5�-GGA TAG GAC CTC AAG ACG CAT GTC TTC T-3�.

��

��

� �� 10^-4�10^-5 cfu/mL, �� 10^-6 cfu/mL �� . �� 1:1500 �� 0,001 OD �� 600 nm. � �� DNA, �� , �� 50 fg, �� 10 ��,¹⁹ �� PCR �� Southern blotting �� PRAVMPAR, � �� 5 fg (1 �� ).

�� , �� . � �� uracil DNA glycosidase, �� (�� PCR, �� , ��), �� .

��

� �� , �� . �� , �� , �� . �� (��. 1), �� , �� .

�� Mtb1�2 �� (98,59%, 70 �� 71) �� Mycobacterium tuberculosis complex (��. 1), �� M. africanum, �� Mycobacterium tuberculosis complex, �� . �� MAC1�2 �� 82,5% (33 �� 40) �� Mycobacterium avium complex (M. avium MAIS2, M. avium serovar 7, M. avium WP M21, M. paratuberculosis Linda, M. paratuberculosis C286, M. paratuberculosis ATCC 19698). �� M16SU-D2 �� 100% (25 �� 25) �� Mycobacterium avium �� �. intracellulare �� 100% (15 �� 15) �� M. paratuberculosis, �� 7 �� �. gordonae �� 4 �� 13 �. smegmatis �� 10. �� DNA �� 509 ��, �� , � �� PRAVMPAR �� 100% �� M. paratuberculosis.

��

�� 75 �� , �� , 59 (79%) �� (��. 3). �� , 38 �� (50,6%) �� Mycobacterium tuberculosis complex, 7 (9%) �� Mycobacterium avium complex, ��, �� , �� 14 (19%) �� M16SU-D2, �� PRAVMPAR. �� 14 �� M. intracellulare, M. gordonae, M. smegmatis (��. 2). �� 35 Ziehl-Nielsen-�� , �� 31 (86%) �� PCR-��, �� 39 �� PCR-�� (��. 3). �� , �� , �� , �� , 4 �� Ziehl-Nielsen-�� PCR. �� .

�� 25 �� , �� , 6 (24%) �� (��. 3), �� Mycobacterium tuberculosis complex. ��, � �� Ziehl-Nielsen �� .

�� 20 �� , �� Crohn, �� 7 (35%) �� DNA �� MAC1�2 �� M16SU-D2. �� , 2 (10%) �� Mycobacterium avium complex, ��, �� , �� 5 (25%) �� M16SU-D2 (��. 3). �� 9 �� PRAVMPAR, �� M. paratuberculosis. �� (Ziehl-Nielsen �� ) �� (��. 3).

�� (��. 3) �� 30 �� , �� 22 �� (BAL) �� Mycobacterium tuberculosis, �� PCR. � �� 4 �� , �� Mycobacterium a vium complex. � Ziehl-Nielsen �� 16 �� 22 ��. �� PCR, �� Ziehl-Nielsen �� 6 �� 10 �� , �� 22 �� , �� . ��, �� 4 �� 4 �� 6 �� (�� 6 �� Crohn), � PCR �� Mycobacterium tuberculosis complex.

��

�� , � �� , �� PCR �� .^25�29 �� , �� Southern blotting � �� DNA ��. �� , ��, �� Mycobacterium tuberculosis complex � �� Mycobacterium avium intracellulare complex, �� . �� , �� ,^31�33 �� PCR, �� -��, �� , �� Ziehl-Nielsen.^14,27 ��, �� , � �� .

�� , � �� , �� , �� . � �� -��, �� , �� , �� , �� 12�24 ��. � ��, ��, �� 16S rRNA, �� PRAVMPAR �� , �� M. intracellulare, M. avium complex, M. smegmatis �� M. gordonae. ��, �� , �� (��, ��, �� Crohn) �� .

�� -�� , �� . �� PCR �� IS6110, �� Mycobacterium tuberculosis complex.^34�36 �� IS6110 �� ,^30,38 �� Mycobacterium tuberculosis complex.^38�40 �� IS6110 �� .³⁸ � �� IS1110 �� Mycobacterium avium complex �� , �� , �� . �� ,⁴¹ �� , �� , �� .⁴² �� Mycobacterium avium complex, �� 16S rRNA,¹⁹ �� M. paratuberculosis, �� , �� IS900 (��. 4), � �� .⁴³ � �� -��. �� , �� , �� M. gordonae �� M. smegmatis. �� IS900 �� 16S rRNA, � �� , �� M. avium �� M. intracellulare. �� MAC1�2 �� 8 �� 15 �� M. paratuberculosis (��. 1), �� IS1110 �� . ��, �� M16SU-D2. �� 10 �� 13 �� M. smegmatis �� 4 �� 7 M. gordonae. ��, �� , �� . � �� .

�� PCR � �� DNA ��, �� . �� PCR, �� Ziehl-Nielsen, � �� . �� . � �� ,^44,45 � �� , �� , �� DNA �� . � �� , ��, �� .

�� , �� ,�� . � �� , �� . �� , �� , �� , �� DNA (�� ) �� .⁴⁶

�� , �� ,^47,48 �� (carry over effect). �� , � �� (HPLC) �� UDG, �� , �� .

� �� , ��, �� (�� ) �� DNA. �� , �� , �� , DNA, �� .⁴⁹ �� , �� , � �� DNA �� .

�� , � �� , �� PCR, �� , �� Ziehl-Nielsen ��, �� , �� .

�� , �� . ��, �� Ziehl-Nielsen-��, �� PCR-��. �� 4 �� (��. 3, �� ), �� Ziehl-Nielsen-��, �� PCR-��. � �� PCR �� , �� , �� PCR (��. 3). � �� Ziehl-Nielsen �� PCR �� PCR-�� Mycobacterium spp.

��, �� Ziehl-Nielsen �� , �� Mycobacterium tuberculosis complex. �� , � Ziehl-Nielsen �� 22 �� 37 PCR-�� (��. 3), �� 33 Mycobacterium tuberculosis complex PCR-�� .

��, �� . �� , �� 30% �� , �� , �� (Ziehl-Nielsen, ��, PCR). �� DNA � �� . � �� , �� , �� , �� , �� , �� . ��, �� , �� PCR-�� .

�� PCR �� , �� Mycobacterium tuberculosis complex. �� (��. 3) �� Mycobacterium avium complex �� Mycobacterium paratuberculosis. �� , �� , �� , �� M. paratuberculosis. �� Mycobacterium avium complex, � �� , � �� ^50,51 �� .

�� , �� , 6 �� . �� , �� , �� . �� Mycobacterium avium complex, �� DNA �� Mycobacterium tuberculosis complex �� ,^45,52 �� , �� ,^5,53�55 �� .^56,57 ��, �� , �� , �� , �� , �� .

�� , �� Mycobacterium avium complex �� , �� , ��, ��, �� 6 �� Mycobacterium tuberculosis complex. �� , �� , �� ,�� . �� , �� , �� 6 ��, �� , �� 8 �� (��, �� , ��). ��, �� 17 ��, �� , �� . �� , �� , �� . �� , ��, �� .⁴³

�� (��. 4) �� Crohn. �� 1932, �� Boril Crohn �� , �� , � �� ,58 �� M. paratuberculosis �� , �� .^59,60

�� , �� M. paratuberculosis (��. 3, �� ) �� 35% �� Crohn. �� , �� 4 �� 6 �� . �� , �� PCR �� . �� , �� , �� . �� PCR �� Crohn, �� . � ��, 15 �� , �� (�� ) �� , �� 6 �� . �� , �� , �� , �� .⁶¹

��, � �� , �� , �� . � �� , �� . � ��, ��, �� , �� Crohn, �� , �� .

* � �� (��)

��

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