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25(1): 39-44 3-2003 T¹p chÝ Sinh häc Gi¸m ®Þnh loµi s¸n l¸ phæi Paragonimus heterotremus Chen et Hsia, 1964 ë viÖt nam b»ng ph−¬ng ph¸p sinh häc ph©n tö Lª thanh hßa, nguyÔn bÝch nga ViÖn C«ng nghÖ sinh häc NguyÔn V¨n §Ò, Lª §×nh C«ng ViÖn Sèt rÐt, Ký sinh trïng vµ C«n trïng trung −¬ng ®Æng tÊt thÕ ViÖn Sinh th¸i vµ Tµi nguyªn sinh vËt HiÖn nay, trªn thÕ giíi cã kho¶ng 50 loµi s¸n l¸ phæi ®* ®−îc x¸c ®Þnh thuéc gièng Paragonimus Braun, 1899 (tªn ®ång nghÜa: Paragonimus Chen, 1963) vµ gièng cã quan hÖ hä hµng víi Paragonimus lµ Euparagonimus Chen, 1962. C¸c loµi s¸n l¸ phæi cã sù ph©n bè kh¸ réng, gåm ch©u ¸ (bao gåm ë phÝa §«ng, tõ Pakistan ®Õn §«ng Nam n−íc Nga, ë phÝa Nam, tõ Xri Lanka ®Õn In®«nªxia vµ Papua Niu Ghinª), ch©u Mü (bao gåm Cana®a ë phÝa B¾c, Brazin vµ Pªru ë Nam Mü) vµ toµn bé ch©u Phi [3]. Nh÷ng tr−êng hîp ng−êi bÞ nhiÔm s¸n l¸ phæi thuéc gièng Paragonimus ®−îc x¸c nhËn t¹i ViÖt Nam vµo n¨m 1993 ë S×n Hå (Lai Ch©u) vµ nguyªn nh©n g©y bÖnh lµ Paragonimus heterotremus [4, 6]. MÆc dï P. heterotremus ®−îc coi lµ nguyªn nh©n g©y bÖnh s¸n l¸ phæi (paragonimiasis) ë ViÖt Nam, nh−ng viÖc x¸c ®Þnh chØ d−¹ vµo h×nh th¸i häc vµ ®Æc tÝnh g©y bÖnh trªn ®éng vËt thÝ nghiÖm lµ chÝnh. VÊn ®Ò ®Æt ra lµ liÖu loµi Paragonimus sp. ®−îc x¸c ®Þnh b»ng h×nh th¸i häc nãi trªn cã ph¶i lµ P. heterotremus hay kh«ng? V× vËy, viÖc gi¸m ®Þnh chÝnh x¸c ®Ó cã kÕt luËn cuèi cïng vÒ loµi nµy lµ cÇn thiÕt ®Ó chÝnh thøc c«ng bè nÕu thùc sù cã Ýt nhÊt sù tån t¹i cña P. heterotremus ë ViÖt Nam. C¸c kÕt qu¶ gi¸m ®Þnh nµy sÏ t¹o c¬ së khoa häc tiÕp theo cho viÖc gi¸m ®Þnh nh÷ng loµi Paragonimus kh¸c (nÕu cã) vµ c¸c chñng kh¸c nhau cña P. heterotremus. C¸c ph−¬ng ph¸p ph©n lo¹i gi¸m ®Þnh sinh vËt dùa trªn chØ thÞ ph©n tö ADN ®* ®−îc ph¸t triÓn vµ øng dông réng r*i, víi nguyªn lý dùa vµo c¸c ®Æc ®iÓm thay ®æi kiÓu gien ë møc ®é ph©n tö thay cho ph©n biÖt thay ®æi h×nh th¸i (kiÓu h×nh) [2, 7]. §é tin cËy cña ph−¬ng ph¸p ph©n tö rÊt cao, ngay c¶ khi ch−a cã biÕn ®æi kiÓu h×nh, cÇn rÊt Ýt mÉu vËt, vµ hoµn toµn kh«ng phô thuéc vµo c¸c giai ®o¹n ph¸t triÓn c¸ thÓ cña sinh vËt. Do vËy, viÖc gi¸m ®Þnh ph©n tö rÊt phï hîp vµ ®−îc øng dông ®Ó ph©n biÖt c¸c loµi ký sinh trïng, v× ®Æc ®iÓm h×nh th¸i cña chóng khã nhËn biÕt h¬n ®éng vËt bËc cao [5]. HiÖn nay, toµn bé chuçi ADN cña hÖ gien ty thÓ (mitochondrial DNA - mtDNA) cña nhiÒu loµi s¸n dÑt, trong ®ã cã s¸n l¸ phæi (loµi P. westermani, Ng©n hµng Gen: AF219379) ®* ®−îc x¸c ®Þnh, còng nh− cã nhiÒu chuçi nucleotit cña nhiÒu loµi Paragonimus kh¸c, t¹o ®iÒu kiÖn thuËn lîi cho gi¸m ®Þnh ph©n tö [1, 2]. Trong nghiªn cøu nµy, chóng t«i ®* sö dông ph−¬ng ph¸p gi¸m ®Þnh loµi cã ®é tin cËy cao nhÊt hiÖn nay trªn c¬ së tr×nh tù gien ty thÓ vµ chÝnh thøc x¸c nhËn Ýt nhÊt cã mét loµi lµ Paragonimus heterotremus Chen et Hsia, 1964, tån t¹i vµ g©y bÖnh ë ViÖt nam, kh¼ng ®Þnh viÖc c«ng bè tr−íc ®©y vÒ loµi nµy cña Kino vµ cs [4]. I. ph−¬ng ph¸p nghiªn cøu 1. MÉu vËt s¸n l¸ phæi MÉu vËt s¸n l¸ phæi tr−ëng thµnh ®−îc thu nhËn tõ hai tØnh Lai Ch©u (ký hiÖu PLCVN) vµ S¬n La (PSLVN). §©y lµ loµi mµ qua kiÓm tra b»ng ph−¬ng ph¸p h×nh th¸i häc ®−îc c«ng bè lµ 39 Paragonimus heterotremus [4]. C¸c mÉu vËt ®Òu ®−îc b¶o qu¶n trong cån 70o, cÊt gi÷ ë nhiÖt ®é -20oC, cho ®Õn khi sö dông. 35 chu kú tiÕp theo: 94oC - 1 phót, 52oC - 1 phót vµ 72oC - 1 phót, chu kú cuèi kÐo dµi 10 phót ë 72oC. 2. Chän chuçi gien ®Ó so s¸nh 5. Gi¶i tr×nh tr×nh tù vµ xö lý sè liÖu Cho ®Õn nay, ngoµi toµn bé hÖ gien ty thÓ (mtDNA) cña Paragonimus westermani (Ng©n hµng Gen, AF219379), c¸c chuçi nucleotit kh¸c cña nhiÒu loµi Paragonimus kh¸c nhau ®Òu lµ cña gien cytochrome oxidase 1 (cox1) vµ chóng ®−îc thu nhËn b»ng ph¶n øng nh©n b¶n gien PCR (polymerase chain reaction). Chóng t«i chän gien cox1 v× gien nµy cã ®é b¶o tån rÊt cao trong hÖ gien ty thÓ [7] ®Ó lµm sè liÖu so s¸nh trong gi¸m ®Þnh loµi Paragonimus sp. cña ViÖt nam. S¶n phÈm PCR ®−îc tinh chÕ b»ng bé ho¸ chÊt QIAquick PCR Purification Kit (QIAGEN Inc.) vµ ®−îc gi¶i tr×nh tù trùc tiÕp hoÆc dßng ho¸ vµo vect¬ pCR2.1 cña bé ho¸ chÊt TAcloning Kit (Invitrogen Inc.) vµ chän läc khuÈn l¹c theo ph−¬ng ph¸p kh¸ng sinh vµ chØ thÞ mµu. T¸ch chiÕt ADN cña plasmit cã chøa PCR ®−îc thùc hiÖn víi bé ho¸ chÊt QIAprep Spin Plasmid Extraction Kit (QIAGEN Inc.). Chuçi ADN ®−îc gi¶i tr×nh tù trªn m¸y ®éng ABI 377 PRISM (Perkin- Elmer) vµ thùc hiÖn víi nhiÒu plasmit t¸i tæ hîp nh»m thu ®−îc kÕt qu¶ chÝnh x¸c. C¸c tr×nh tù t−¬ng øng víi ®o¹n ADN nghiªn cøu tõ mét sè quÇn thÓ thuéc gièng Paragonimus ®* ®−îc c«ng bè [2] vµ ®¨ng ký t¹i Ng©n hµng Gien, ®−îc sö dông ®Ó so s¸nh ®èi chiÕu. S¾p xÕp, ®èi chiÕu tr×nh tù t−¬ng øng cña tõng ®o¹n gien b»ng hÖ ch−¬ng tr×nh m¸y tÝnh AsemblyLIGN 1.9 vµ MacVector 6.5.3 (Oxford Molecular Inc.). Tr×nh tù axit amin ®−îc dÞch m* theo b¶ng m* di truyÒn mt-DNA sè 21 cña s¸n dÑt (platyhelminth mitochondrial code) giíi thiÖu trong Ng©n hµng Gen. Ph¶ hÖ s¬ bé ®−îc xö lý b»ng ch−¬ng tr×nh phô trong hÖ MacVector 6.5.3. 3. T¸ch chiÕt ADN tæng sè ADN tæng sè ®−îc t¸ch chiÕt b»ng bé ho¸ chÊt DNeasy Tissue Kit (QIAGEN Inc.) theo quy tr×nh cña nhµ s¶n xuÊt. M« t¶ rót gän nh− sau: mÉu vËt b¶o qu¶n trong cån 70o ®−îc lÊy ra vµ cho cån bay h¬i hÕt, sau ®ã röa nhiÒu lÇn trong PBS. MÉu vËt ®−îc nghiÒn kü vµ xö lý víi c¸c dung m«i cña Kit, råi hÊp phô lªn mµng vµ ly chiÕt ADN theo quy tr×nh t¸ch chiÕt. Hµm l−îng ADN sö dông cho mçi ph¶n øng PCR (50 microlit) lµ kho¶ng 150 nanogam. 4. Måi (primer) vµ tiÕn hµnh ph¶n øng PCR CÆp måi ®−îc thiÕt kÕ ®Ó dïng trong nh©n b¶n ADN ®Ých b»ng ph¶n øng PCR dùa trªn c¬ së c¸c tr×nh tù b¶o tån cña gien cox1. Måi xu«i JB3F: 5’ TTTTTTGGGCATCCTGAGGTTT AT3’ vµ måi ng−îc JB4.5R: 5'TAAAGAAAGA ACATAATGAAAATG3'. ADN ®Ých ®* ®−îc nh©n b¶n b»ng PCR tiªu chuÈn víi bé ho¸ chÊt PCR Master Mix Kit (Promega). Chu tr×nh nhiÖt cña PCR trªn m¸y Perkin-Elmer (Perkin-Elmer Inc., Mü) gåm c¸c b−íc nh− sau: 94oC - 5 phót, 6. §Þa ®iÓm tiÕn hµnh gi¸m ®Þnh C«ng viÖc gi¸m ®Þnh ph©n tö vµ gi¶i tr×nh tù ®−îc tiÕn hµnh t¹i phßng thÝ nghiÖm Ký sinh trïng häc ph©n tö thuéc ViÖn nghiªn cøu Y häc Queensland, «xtr©ylia. II. KÕt qu¶ vµ th¶o luËn 1. §èi chiÕu ph©n tö vµ so s¸nh ph©n ®o¹n gien cox1 A. PhetChina PhetThai PSLVN1 PSLVN2 PLCVN1 PLCVN3 PLCVN4 Pbangkok_H P_harinasu P_siamensi Piloktsuen Pmacrorchi 40 : : : : : : : : : : : : * 20 * 40 * 60 * 80 * TTTAATTTTGCCTGGATTTGGTGTTGTGAGACATATCTGCATGACTTTGACTAATAAAGATTCTTTGTTCGGTTATTATGGCTTGGTTTTTGCC .............................................................................................. .....................................................................T........................ .....................................................................T.......................T .....................................................................T.......................T .....................................................................T........C............... .....................................................................T........................ .C.G...C.A..C..G.....G........T..C..T..........................GC....T...........G.....G..C..T ...G.....A..G........A.....A..T.....T..........................GC....T.....C.....G.....G..C..T ...G...........T.....GA.............T..T...........G.....T......C....T...........G..A..G.....G .........A..A........GA.............T..T......C.A..............G.....T...........A.....G.....T ............A..G....................T..T.....G..A........C..C........T...........T..A......... : : : : : : : : : : : : 94 94 94 94 94 94 94 94 94 94 94 94 PMexEcuado : ...G........C........G.....T........T..T.................T...........T...........G..A........G : PohiraiKin : .........A..A........GA.............T..T......C.A..............G.....T...........A.....G.....T : PohiraiTan : .........A..A........GA.............T..T......C.A..............G.....T..C........A.....G.....T : 94 94 94 PhetChina PhetThai PSLVN1 PSLVN2 PLCVN1 PLCVN3 PLCVN4 Pbangkok_H P_harinasu P_siamensi Piloktsuen Pmacrorchi PMexEcuado PohiraiKin PohiraiTan : : : : : : : : : : : : : : : 100 * 120 * 140 * 160 * 180 ATGGGGGCTATTGTGTGTTTGGGGAGGGTTGTTTGAGCGCACCATATGTTTATGGTTGGTTTAGATGTCAAGACTGCTGTTTTTTTTAGTTCTG .............................................................................................. ....................................................................T......................... ............................................C.......................T......................... ............................................C................................................. ............................................C................................................. ............................................C................................................. .....A..G.....T........T..T.....G..G..T..T....................G.....T.....C..G..G........A.... ........G.....T........T..T.....G.....C..T..........................T.....C..G..G........A.... .....A.................A..T.....G..G.....T....................G.....T...........G............. ........G..............A..T.....C..G..C.......................G.....T........A..A.....C..A.... ..............T...........T..G.....G.....T..................C.T.....T....................C.... ...........C..A.....A..A..............A..T....................G.....T........C..............G. ........G..............A..T.....C..G..C.......................G.....T........A..A.....C..A.... ........G..............A..T.....C..G..C.......................G.....T........A..A.....C..A.... : : : : : : : : : : : : : : : 188 188 188 188 188 188 188 188 188 188 188 188 188 188 188 PhetChina PhetThai PSLVN1 PSLVN2 PLCVN1 PLCVN3 PLCVN4 Pbangkok_H P_harinasu P_siamensi Piloktsuen Pmacrorchi PMexEcuado PohiraiKin PohiraiTan : : : : : : : : : : : : : : : * 200 * 220 * 240 * 260 * 280 TTACTGGGGTGATTGGGATTCCCACAGGGATTAAGGTTTTTTCTTGGTTGTTTATGTTGGGGGGGACTCGTTTACGGTTTTGAGATCCGGTGGT .............................................................................................. .............................................................................................. .............................................................................................. .............................................................................................. ......................T....................................................................... .............C................................................................................ ....G........A..T..C..G.........................................T........G..............T..AA. .............A..T..C..G....................C.................T..C........G..............T..AA. .C..C.....T........A..T..G..T.................A..A..C.....A..T...G.......G..TC....G.....T..CT. ....G........A..T.....T..........................A........A.....C......C..........G.....T..AA. ....G...........T.....T..G..T...........................C.A.....A.....C...........G.....C..AA. .......T........T.....T..G................................A..T..T........G..A..C........T..... ....G........A..T.....T..........................A........A.....T......C..........G.....T..AA. ....G........A..T.....T..........................A........A.....T......C..........G.....T..AA. : : : : : : : : : : : : : : : 282 282 282 282 282 282 282 282 282 282 282 282 282 282 282 PhetChina PhetThai PSLVN1 PSLVN2 PLCVN1 PLCVN3 PLCVN4 Pbangkok_H P_harinasu P_siamensi Piloktsuen Pmacrorchi PMexEcuado PohiraiKin PohiraiTan : : : : : : : : : : : : : : : * 300 * 320 * 340 * 360 * TTGGTGAATTTTAGGCTTTATTTTTCTTTTTACTATTGGTGGTGTAACTGGGATTATTTTGTCTTCTTCTATTTTGGATAGTCTGTTACATGAT .............................................................................................. .........C........................................................C........................... .............................................................................................. .............................................................................................. .............................................................................................. ....................................................................................A......... A...........G..G.....C.....G........C..G.....G..............A...........C.....C...T....G.....C A...........G..G...........G...........G.....G.....A..............................T....G...... G.....G...C.T..T..C.....CT.G........A........G..C..T...G.......C.....A..A.........T....G...... A..A......C.T..G.....C...T.G.....A..A..G...........A.....C....................C...T.A..G.....C ...A........G..A....................A.....G..G..G..........................A......T....G...... G..A..G........G.................C..A......................................A..C...T..........C A..A......C.T..G.....C...T.G.....A..A..G...........A.....C....................C...T.A..G.....C A..A......C.T..G.....C...T.G.....A..A..G...........A.....C....................C...T.A..G.....C : : : : : : : : : : : : : : : 376 376 376 376 376 376 376 376 376 376 376 376 376 376 376 B. PhetChina : PhetThai : PSLVN1 : PSLVN2 : PLCVN1 : PLCVN3 : PLCVN4 : Pbangkok : P_harinasu : P_siamensi : P_iloktsuen: P_macrorchi: P_mexicanus: P_ohirai1 : P_ohirai2 : P_sado : * 20 * 40 * 60 * LILPGFGVVSHICMTLTNNDSLFGYYGLVFAMGAIVCLGSVVWAHHMFMVGLDVKTAVFFSSVTGVIGIPT ....................................................................... ....................................................................... ....................................................................... ....................................................................... ....................................................................... ....................................................................... ....................................................................... ....................................................................... .......I............................................................... .......I............................................................... ....................................................................... ....................................................................... .......I............................................................... .......I............................................................... .......I............................................................... PhetChina PhetThai PSLVN1 PSLVN2 80 * 100 * 120 GIKVFSWLFMLGGTRLRFWDPVVWWILGFIFLFTIGGVTGIILSSSILDSLLHDTWFV .......................................................... .......................................................... .......................................................... : : : : : : : : : : : : : : : : : : : : : : : : 71 71 71 71 71 71 71 71 71 71 71 71 71 71 71 71 129 129 129 129 41 PLCVN1 : PLCVN3 : PLCVN4 : P_bangkok : P_harinasu : P_siamensis: P_iloktsuen: P_macrorchi: P_mexicanus: P_ohirai1 : P_ohirai2 : P_sadoensis: .......................................................... .......................................................... .......................................................... ......................I................................... ......................I................................... .............A...L....L..................V................ ......................I................................... ......................I................................... .......................................................... ......................I................................... ......................I................................... ......................I................................... : : : : : : : : : : : : 129 129 129 129 129 129 129 129 129 129 129 129 H×nh 1. So s¸nh tr×nh tù nucleotit (A) vµ axit amin (B) cña ®o¹n gien cox1 cña mét sè chñng Paragonimus sp. cña ViÖt Nam víi c¸c chñng P. heterotremus cña Trung Quèc vµ Th¸i Lan, còng nh− cña c¸c loµi kh¸c nhau trªn thÕ giíi Ghi chó: dÊu (.): biÓu thÞ gièng víi tr×nh tù P. heterotremus cña Trung Quèc; sai kh¸c vÒ nucleotit hoÆc axit amin ®−îc thÓ hiÖn b»ng c¸c ch÷ c¸i ký hiÖu cña chóng. PhetChina vµ PhetThai: Paragonimus heterotremus Chen et Hsia, 1964 (chñng Trung Quèc vµ chñng Th¸i Lan); PSLVN: Paragonimus sp. ViÖt Nam (chñng S¬n La); PLCVN: Paragonimus sp. ViÖt Nam (chñng Lai Ch©u); P_bangkok: P. bangkokensis Miyazaki and Vajrasthira, 1967; P_harinasu: P. harinasutai Miyazaki and Vajrasthira, 1968; P_siamensis: P. siamensis Miyazaki and Wykoff, 1965; P_iloktsuen: P. iloktsuenensis Chen, 1940; P_macrorchi: P. macrorchis Chen, 1962; P_mexicanus: P. mexicanus Miyazaki and Ishii, 1968; P_sadoensis: P. sadoensis Miyazaki, Kawashima, Hamajima and Otsuru, 1968. H×nh 1A cho thÊy tr×nh tù nucleotit cña ®o¹n gien cox1 cña Paragonimus heterotremus cña Trung Quèc vµ Th¸i Lan lµ hoµn toµn gièng nhau. C¸c chñng Paragonimus sp. cña ViÖt Nam thu nhËn t¹i S¬n La (ký hiÖu PSLVN1 vµ PSLVN2) chØ sai kh¸c 4 nucleotit vµ c¸c chñng thu nhËn t¹i Lai Ch©u (ký hiÖu PLCVN1, PLCVN3, PLCVN4) chØ sai kh¸c 3-4 nucleotit, cho møc ®é t−¬ng ®ång lµ 99,0-99,2%. Sai kh¸c gi÷a c¸c chñng Paragonimus sp. cña ViÖt Nam víi tr×nh tù cña c¸c loµi kh¸c, vÝ dô víi loµi P. ohirai lµ 55/376, do vËy møc ®é t−¬ng ®ång lµ rÊt thÊp (85.4%); hoÆc víi loµi P. siamensis lµ 62/376, tû lÖ t−¬ng ®ång chØ ®¹t 83,5%. Tr×nh tù axit amin ë tÊt c¶ c¸c chñng cña P. heterotremus cña ViÖt Nam, Trung Quèc vµ Th¸i Lan lµ hoµn toµn gièng nhau. Thµnh phÇn axit amin cña c¸c loµi kh¸c còng kh«ng thay ®æi lín, trõ loµi P. siamensis (5/129, ®¹t 3,9%), chøng tá sù thay ®æi vÒ nucleotit kh«ng hoÆc lµm thay ®æi rÊt Ýt axit amin trong thµnh phÇn cña ®o¹n gien cox1 nghiªn cøu (h×nh 1B). loµi ë c¸c ch©u kh¸c nh»m ®¶m b¶o tÝnh kh¸ch quan cña sù ph©n tÝch. Nãi chung, c¸c chñng cïng 1 loµi ®Òu ®−îc ph©n nhãm cïng nhau, vÝ dô loµi P. myiazaki cña NhËt B¶n hay loµi P. skrÞabini. TÊt c¶ c¸c chñng trong loµi Paragonimus sp. cña ViÖt Nam vµ Paragonimus heterotremus cña Trung Quèc vµ Th¸i Lan ®Òu n»m chung trong mét nhãm, trong nh¸nh ph¶ hÖ kh¸c víi mäi loµi kh¸c (h×nh 2). Tû lÖ t−¬ng ®ång vÒ nucleotit còng nh− axit amin cã thÓ coi lµ tuyÖt ®èi cña c¸c chñng trong loµi Paragonimus sp. cña ViÖt Nam (trªn 99%) vµ ph©n nhãm hoµn toµn cïng víi Paragonimus heterotremus cña Trung quèc vµ Th¸i lan, cho phÐp chóng ta kÕt luËn loµi Paragonimus sp. cña ViÖt Nam (chñng Lai Ch©u vµ S¬n La) lµ Paragonimus heterotremus qua so s¸nh ph©n tÝch tr×nh tù vµ s¾p xÕp ph¶ hÖ. III. KÕt luËn 2. S¬ bé xem xÐt quan hÖ loµi cña P. heterotremus cña ViÖt Nam 1. Gi¸m ®Þnh sinh häc ph©n tö chÝnh thøc cho thÊy c¸c mÉu s¸n l¸ phæi Paragonimus sp. cña ViÖt Nam (chñng Lai Ch©u vµ S¬n La) lµ Paragonimus heterotremus Chen et Hsia, 1964. H×nh 2 biÓu thÞ s¬ bé ph¶ hÖ vµ ph©n nhãm cña c¸c loµi vµ c¸c chñng trong gièng Paragonimus. C¸c loµi chän läc ®Ó ph©n tÝch ph¶ hÖ bao gåm nh÷ng loµi ë ch©u ¸ vµ nhiÒu 2. Paragonimus heterotremus cña ViÖt Nam cã møc ®é t−¬ng ®ång ph©n tö rÊt cao víi P. heterotremus cña Trung Quèc vµ Th¸i Lan (trªn 99,0% vÒ nucleotit vµ 100% vÒ axit amin). 42 Pxiangshanensis.txt Pmenglaensis.txt 0.056 0.021 0.003 0.005 PbangkokHainan.txt 0.033 0.028 Pharinasutai.txt 0.033 PwThai.txt 0.040 0.009 PwleytePhilippine.txt 0.024 0.036 0.005 0.062 PwMingqing.txt 0.002 0.005 0.006 0.001 0.017 0.001 0.004 0.004 PwMieJpan.txt PwKorea.txt PwHyogo.txt PwAmak3n.txt Psiamensis.txt 0.065 EupNanjing2.txt 0.003 EupNanjing1.txt 0.067 0.009 0.016 0.007 BigAnhui2.txt BigAnhui.txt 0.001 Ppaish2.txt 0.002 Ppaish1.txt 0.075 PsadoensisJp.txt 0.000 PohiraiKinosakiJp.txt 0.001 0.001 0.010 0.064 0.045 PLCVN3.txt PLCVN1.txt 0.002 0.002 0.000 PSL383.txt 0.000 0.007 0.005 0.002 0.035 PhetThai.txt PhetChina.txt PSL331.txt 0.010 0.006 0.006 Piloktsuenensis.txt PMexEcua.txt PLCVN4.txt 0.005 0.000 0.005 0.010 PohiraiTanegJp.txt 0.002 0.066 PSL1.txt 0.240 0.058 PsYunnan1.txt 0.039 0.002 0.001 0.003 0.013 0.003 0.007 0.009 0.012 0.006 0.004 Phokuoensis2.txt 0.006 0.010 0.003 0.003 0.000 0.002 0.002 PsHubei1.txt PsJapan(Ken).txt PsGuangdong.txt PsGuang1.txt 0.028 0.000 0.003 0.008 0.002 PsSichuan.txt 0.002 0.017 0.000 0.001 Parag-like.txt Pmacrorchis.txt Phokuoensis1.txt PmiyaOK.txt PsJian1.txt PmiyaJianou.txt PsJanou.txt PmiyaShizu.txt PmiyaNanko.txt PmiyaHiwas.txt H×nh 2. S¬ bé ph¶ hÖ dùa trªn tr×nh tù nucleotit cña ®o¹n gien cox1 cña mét sè chñng Paragonimus sp. cña ViÖt Nam víi c¸c chñng P. heterotremus cña Trung Quèc vµ Th¸i Lan, vµ c¸c loµi kh¸c nhau trªn thÕ giíi. Paragonimus sp. cña ViÖt Nam ®−îc liÖt kª vµo cïng nhãm víi P. heterotremus cña Trung Quèc vµ Th¸i Lan 43 3. S¬ bé ph©n ®Þnh ph¶ hÖ cho thÊy P. heterotremus cña ViÖt Nam lµ hoµn toµn cïng nhãm víi P. heterotremus cña Trung Quèc vµ Th¸i Lan, nh−ng kh¸c xa víi c¸c loµi Paragonimus kh¸c. Tµi liÖu tham kh¶o 1. Blair D., 1993: Acta Trop., 53: 227-289. 2. Blair D. et al.,1998: Proceedings of the 9th International Congress of Parasitology. Chiba, Japan: 643-647. 3. Blair D., Xu Z. B. and Agatsuma T., 1999: Adv. Parasitol., 42: 113-222. 4. Kino H. et al., 1995: Jpn. J. Parasitol., 44: 470-472. 5. Mc Manus D. P. and Bowles J., 1996: Int. J. Parasitol., 26: 687-704. 6. NguyÔn ThÞ Lª, §Æng TÊt ThÕ, Ph¹m Ngäc Doanh, 1997: T¹p chÝ Y häc ViÖt Nam, 2: 35- 40. 7. Lª Thanh Hßa vµ Mc Manus D. P., 2001: TËp san Sinh häc - Héi nghÞ quèc tÕ vÒ sinh häc. Hµ Néi 2-4/7/2001: 101-108. molecular identification of paragonimus heterotremus Chen et Hsia, 1964 in vietnam Le Thanh Hoa, nguyen bich nga, Nguyen Van De, le dinh cong, §ang Tat The summary A region of 376 nucleotides of the mitochondrial-encoded cox1 gene from 6 samples of Paragonimus sp. collected in the Laichau and Sonla provinces was amplified by polymerase chain reaction (PCR) and comparatively aligned with the known corresponding sequences of Paragonimus heterotremus (geographical origin of China and Thailand) and representative species of the Paragonimus genus (GenBank and published data). Molecular-based analysis revealed that Paragonimus sp. of Vietnam is Paragonimus heterotremus Chen et Hsia, 1964, showing high nucleotide similarity to that of China and Thailand strains (over 99%). Phylogenetic analysis uniquely groups the Paragonimus sp. of Vietnam with the Paragonimus heterotremus of China and Thailand. Ngµy nhËn bµi: 16-11-2001 44