Detection of infectious canine hepatitis virus by TaqMan real-time PCR method

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(1)³¤¥Èó. * .* a. (2004) 44 4 Korean J Vet Res(2004) 44(4) : 655~662. 5BR.BO *1$3»ö~RB*9*:

(2) Ê~¦Â. {B'ÁRÒÏÁêÁ;ê^Á` âÁRÒÆÁRã9Áj&+ *§L >~ Ún*W \² (²Òß: 2004j 7ú 20¢). Detection of infectious canine hepatitis virus by TaqMan real-time PCR method Hye-young Wang, Jae-yong Choi, Mi-jin Lee, Jin-ho Park, Mae-rim Cho, Jae-cheol Han, Kyoung-seong Choi and Joon-seok Chae* Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea (Accepted= July 20, 2004) Abstract : The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines. Key words : infectious canine hepatitis, canine adenovirus, E3 ORFA gene, real-time PCR. * . º ¦VÏö ~ " *2B [2, 25]. î÷b ¦V ²B Bf Öº CAV-1~ "º ¶ j ~² >, virus¢ 6-9 Bú ÿn Ú VJ [2, 26, 27]. j;¾ ;6ïj Û~ Þêö «B CAV-1f *' :Ê.Ãj FB êö, *, Ë Ò ." ?f Ú~ ç^

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(4) ¢ ¶çÊº ß û ® [1, 13, 29, 30]. ªç÷Ò' ¦Ò ²Ò~ ßûbº .7 .²6> ~ 6², j;ç' .²6 VË, Ò prothrombin time~ Ë " ?f disseminated intravascular. B *"W *" f ö ~~ FB>º : ÊW î÷. ~ ¢« º ~ ³ö ³~ ç ã ~ ; Ú ç & :Ê. f *"W *"j FB ~ º ^V 6"Ãj FB~º ©b rJ^ ®. 7 f *"W. Ö ;~ 6"B ÿb"~ 7/¾ J"B Bb $. (infectious canine hepatitis, ICH) canine adenovirus(CAV) type 1 [1, 2, 13, 18, 26]. DNA virus CAV Adenoviridae Mastadeno virus , 70 - 80 nm 20 [1, 11, 17]. CAV type 1 (CAV-1) , type 2 (CAV-2) [1, 13, 18]. CAV-1 ,. ¢^f 2003jê *§&L Ún*W\² .\j~ ¢¦æöbR Úrr(CNU-BSRI, No. 2003-5). *Corresponding author: Joon-seok Chae Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea [Tel: +82-63-270-3881, Fax: +82-63-270-3778, E-mail: [email protected]]. 655.

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(7) Ê~ ¦Â. ¢ F;~& Ö" .ç>º Ã Öb ~ V& >ê f ¢ B·~&. $ j *~ ¢ B·~&. " º '' f B ö¶~ ·öö ¦O~&. 7Î²ê>w PCR~ >wbº C 20 µl Úö SuperTaq 10Ü Reaction buffer I" II, 2.5 mM~ dNTP, '' 10 pmol~ HeT-Ff -R primer, 2.5 U~ Taq DNA polymerase(Super Bio Co., Ltd, Korea), Ò B ºÂB 100 ng~ template DNA¢ Î&~& . >wbº predenaturationj 95 CöB 5ª* 1² >¯ êö, denaturation" annealing, polymerizationj '' 95 Cö B 1ª*, 56 CöB 1ª*, 72 CöB 1ª* 40² >¯ ~& . Ò 72 CöB 10ª* post-extensionj ~& . F*¶ Ã Vº PTC-200 thermocycler(MJ Research, Waltham, USA)j ÒÏ~& . Ã B F*¶ Öbf 2% agarose gel çöB *V' ÿj >¯~ ªÒ~&, ethidium bromide(EtBr, Sigma, St Louis, MO) DNA~ "ïj ~& . PCR Ã ¦f Ã Öb~ Vº still video documentation system(Gel Doc 2000, Bio Rad, USA)j Ï~ &V ~&, DNA size markerº 100 bp DNA ladder (Genepia, Korea) ÒÏ~& . F*¶ 5 "8B ªC Ã B F*¶º QIAquick PCR purification Kit (Qiagen, Germany)¢ ÒÏ~ PCR Öbj ºÂ~&. . ºÂB DNA¢ pGEM-T easy(Promega, USA) vector ö ÖV . $ PCR ¦Â R&"¦V Ã Î CAV-1~ E3 ORFA F*¶ $ pGEM-T easy vector¢ Ï~ Ò plasmid DNA¢ » ê, · W &b ~& (Fig. 1). ö ;WB Ò plasmid¢ TOP10F' E. coli competent cells Úö ã«~ ;î*~ V . Ò f F Ræ ampicilline(50 µg/ml)j Ï~ F ê~&b, Ò plasmid DNAº alkaline lysis O» ö &~ plasmid mini prep kit (Qiagen, Germany)¢ Ò Ï~ ºÂ~& . F*¶ "VBf dideoxy chain termination O»ö &~ automatic sequencer (ABI. 657. probe . , , CAV1-E3F (5'E3 ORFA 151 bp CACATGCCACATAAGCGAAA-3') CAV1-E3R(5'CATACGGCATCTGGTGGCAGT-3') primer (Genotech, Korea). , TaqMan PCR CAV1E3P(5'-6FAM-TTTCCAAGGATGGCCTATACATCGCCATAMRA-3') probe . FAM(6-carboxyfluorescein) TAMRA(6-carboxytetramethylrhodamine) reporter quencher dye .. o. o. o. o. o. TM. Fig. 1. Plasmid map of pGEM-Teasy/E3ORFA with infectious canine hepatitis E3 ORFA gene fragment.. f ABI Prism ¢ Ï~ ª C~& $ ªCB F*¶ "VBf BLAST ¢ Ï~ jv~&. prism 377, Applied Biosystem, USA) Sequence analysis software(version. 2.1.1) . , network service ..

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(10) * ~ ¦Â "6W ¦Âj * ¢> PCR »" TaqMan * PCR »"~ "6Wj jv ªC~V *~ B »B pGEM T easy/CAV1 E3 ORFA plasmid (Fig. 1) DNA¢ 10 ¦V 10 copy ræ ³'b 10VO C ê, PCR~ Template DNA '' ÒÏ~& . Ö" ¢ > PCRj Ï ê»öBº 10 copy~ plasmid DNA ¢ Î& ¦ªræ CAV-1 E3 ORFA F*¶& Ã > î (Fig. 4A). Þ, * PCRj Ï ê»öB º 1 copyræ E3 ORFA F*¶& Ã >îº, 10 copy¢ Î& >wöBº 15 cycle¦V ;7ï²¢ 6æ~ E3 ORFA F*¶& Ã >î, 1 copy¢ Î & >wöBº 33 cycle¦V E3 ORFA F*¶& Ã >º ©j { > ®î (Fig. 4B). V¢B TaqMan. * PCRj Ï ê» ¢> PCRj Ï ê » £ 1,000V ;ê "6W ¸f ©j r > ®î . ªçòöB~ F*¶ ¦Â ÎNW ¢> ÿb÷öb¦V ICH ~>Ú ~ÖB 61î Ò~ .öB ºÂB DNA¦V CAV-1~ ß F* ¶ ¦Âj ~& . Ö", C 61B~ ò 7 ¢ > PCRj Ï ê»öBº 18B(29.5%)öB, ICH. 9. 0. 3. Fig. 2. Specificity of canine adenovirus type 1 E3 ORFA primers used for conventional. M: 100 bp DNA ladder, lane 1: canine adenovirus type 2 (CAV-2) 1, lane 2: CAV-2 2, lane 3: CAV-2 3, lane 4: Bordetella bronchiseptica 1, lane 5: B. bronchiseptica 2, lane N: canine genomic DNA, lane P: positive control(CAV-1).. Ò ;ç B . F*¶¦V '' ¢ ºÂ êö ¢> PCR ". * j >¯ Ö" ¢>' PCR ". * ÎvöB ò ß'b F*¶ Þòj Ã > ®îb &b ÒÏ öÚ öBº ''~ ÎvöB Ã ~æ p~. CDV, CCV, CPV, leptospira, parainfluenza DNA TaqMan PCR , TaqMan PCR CAV-1 ICH , DNA PCR (Fig. 3).. 9. Fig. 3. Specificity of canine adenovirus type 1 E3 ORFA primers used for conventional and TaqMan real-time PCR. A: Specificity tested by conventional PCR. M: 100 bp DNA ladder, lane 1: infectious canine hepatitis virus (ICHV), lane 2: canine distemper virus (CDV), lane 3: canine coronavirus (CCV), lane 4: canine parvovirus (CPV), lane 5: leptospira, lane 6: parainfluenza, lane 7: canine genomic DNA, lane 8: water control. B: specificity test by TaqMan real-time PCR..

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(12) Ê~ ¦Â. 659. Fig. 4. Comparison of sensitivity of conventional PCR and Real-time TaqMan PCR for detection of infectious canine hepatitis virus. A: Amplification of CAV-1 E3 ORFA gene using conventional PCR with 10-fold serial dilution of plasmid DNA template. Serial dilutions were made using adenovirus plasmid DNA by cloning. A minimum of 103 copy of DNA could be detected after 40 cycles. B: Standardization of Adenovirus E3 ORFA gene in the TaqMan real-time PCR assay. Serial dilutions were made using Adenovirus plasmid DNA by cloning. A minimum of 1 copy of DNA could be detected after 40 cycles. The intensity of fluorescence is given on the y axis (∆Rn=reporter signal [FAM]/passive reference signal [TAMRA]). Table 1. Comparison of conventional and TaqMan realtime PCR methods for detection of infectious canine hepatitis virus from canine bloods (P<0.005) Number of Amplified by Amplified by TaqMan blood samples conventional PCR real-time PCR 61. 18(29.5%). 23(37.7%). * j Ï ê»öBº 23B öB F*¶~ Ã &V> î. V¢B. * PCRj Ï ê» ¢> j Ï ê» 8.2% ; ê ê~ ÎNW ¸f ©j r > ®î (P<0.05). $, . òöB ¢> PCR»" TaqMan * PCR»~ CAV-1ö & ¦Â~ "6ê¢ jv ~. . b&, ªç ò¢ &çb þöB PCR ·W >wj ¾æÞ vB~ ò¢ Fê~ ºÂB DNA¢ 10VO ³'b C~ 100 ng/µl ¦V 1 fg/µl ;~& . ¢ &æ '' PCRj >¯~ Ö ", ¢> PCRöBº '' 1 ngöB 100 pgræ, TaqMan. TaqMan PCR (37.7%) CAV-1 E3 ORFA (Table 1). TaqMan ICH PCR. * PCRöBº 10 fgræ CAV-1 E3 ORFA F*¶ & Ã >î (Fig. 5). V¢B Bf îR&æ, &¦ bj &çb ICH ê»ê TaqMan * PCR » ¢> PCR» £ 1,000VöB 10,000V ;ê~ "6W ¸f ©j r > ®î .. V. *"W î÷~ ãÖöº B÷ ¯, ³~ «{ êj Û~ ¯'' ¾~¢ >¯bB î÷b b¢ ²z > ® . ICHf ? *"W ¸f î÷ö &Bº ÷öÚö & ß', ³~² ê > ®º ê» {ã>Ú ® ;{ êö êæ F © . "¾ö BB>Ú jçræ ê Þ&® > ®º PCRj Ï ê»f ß W" "6W, Ò ³W Vö ÒÏ>Úê . Ó' O»¾ z' O»j Ï ê». Î"', :Ê öj ¦Â º ~& ®. ~Æ [4, 5, 15]. ß®, &¦bö Ò~º ÷öÚ~ >& Ôf ãÖ¾ ÷öÚ~ ªÒ& r Ú ãÖöB.

(13) 660. {B'ÁÒÏÁêÁ;ê^Á âÁRÒÆÁã9Áj&+. Fig. 5. Amplification of canine adenovirus type 1 E3 ORFA gene using conventional PCR and real-time TanMan PCR with 10-fold serial dilution of template DNAs from two clinical positive samples (A and B, sample 1; C and D, sample 2). A: A minimum of 100 pg (lane 4) of DNA could be detected by conventional PCR. C: A minimum of 1 ng (Lane 3) of DNA could be detected by conventional PCR. B and D: A minimum of 10 fg (lane 8) of DNA could be detected. The intensity of fluorescence is given on the y axis(∆Rn=reporter signal[FAM]/passive reference signal[TAMRA]). Lane 1, 100 ng/µl; lane2, 10 ng/µl; lane 3, 1 ng/µl; lane 4, 100 pg/µl; lane 5, 10 pg/µl; lane 6, 1 pg/µl; lane 7, 100 fg/µl; lane 8, 10 fg/µl; lane 9, 1 fg/µl; lane N, negative control; lane W, water control.. ê PCRj 'Ï ê» V~ O»j 'Ï ê »ö j~ ú® ¸f ¦ÂW'j ¾æÞ >Ú^ ® [5, 16]. F öBº " º *"W ÷öÚö &~ ¢> PCR" * PCRj Ï ê» B® >Ú J ® [7, 11, 19, 23, 25, 28]. ¾ ICHö & * PCR» jçr æ >Úææº pf ç, z× ÖÒ¾¢ö Bº O» j Ï ICH~ ê» *& > ¯> ®æ p . ICH òj ß'b ê~V *~ ®ö B·B E3 ORFA primer 3f ¢> PCR" * PCR Îvö B CAV-1öò ß'b >wj ~&, &b ÒÏ CDV, CCV, CPV, leptospira, parainfluenzafº * & >wj ~æ p~ . Ö" j, E3 ORFA primer 3j Ï PCR»f ICHf ?f *"W î÷ ö & ê~ ßW Ö ¸ º 6j { > ®îb, ICHö & ¢> 5 * PCR ê»j {ã > ®² >î . Þ, ¢> PCR»f >w Â Ã Öbj agarose gelöB *V'ÿb ªÒ êö EtBr "ïj ~,. UV transilluminator çöB &V "Ú¢ ~º ® Ú ";j ö¢ò >w¦¢ 6; > ®îb¾,. * PCR»f Ã B Öbj Sequence Detector(Ver.. *Îj Ï~ : V Î îV çöB { > ®² >î [11, 19, 23, 28]. V ¢B * PCR»f V~ PCR»öB > ö ¢ò ~º ®Ú ";j Û > ®V r^ö R z ³~² >w¦¢ 2k > ®² >îb, $ Ã >º F*¶& >wö Î&>º probefê 27b >wj ~¢ò ¦ÂF > ® º ßû r^ ö ê~ ßW R ¸jr > ®Æ. [11, 19, 23, 28]. ® þöBº ¢> PCR" * PCR~ ICHö & ¦Â~ "6W" ³W, Ò ÎNWj jv ª C Ö" B'b * PCR»j 'Ï ãÖ öº DNA ºÂ¦V >w «òræ £ 3* ;ê Ú ö ICHö & 6"¦¢ { > ®îV r^ö, V~ PCR» £ .>;ê~ *ò ²º>º ©j r > ®î . V¢B ê~ ³W /® º >º *"W î÷~ ãÖöº V~ PCR» . * PCR» z FÏ~ > ®Æ . $ , ICH~ R&"¦V »B plasmid DNA¢ Ï "6W~ jv ªCöBê, * PCRj Ï ê » ¢> PCRj Ï ê» £ 1,000V ; ê~ "6W ¸ º 6ê r > ®î . $, *§æ~ ÿb÷öb¦V ICH ~>Ú. 1.7, ABI, USA).

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(16) 662. {B'ÁÒÏÁêÁ;ê^Á âÁRÒÆÁã9Áj&+. 9. Craig, E. G. Infectious canine hepatitis and canine acidophilic cell hepatitis. In: Craig. E. G. (Edied.), Infectious Diseases of the Dog and Cat. p. 242-250. Saunders, Philadelphia, 1990. 10. Darai, G., Rosen, A., Delius, H. and Flugel, R. M. Characterization of the DNA of canine adenovirus by restriction enzyme analysis. Intervirology. 1985, 23, 2328. 11. Desjardin, L. E., Chen, Y., Perkins, M. D., Teixeira, L., Cave, M. D. and Eisenach, K. D. Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis. J. Clin. Microbiol. 1998, 36, 1964-1968. 12. Dragulev, B. P., Sira, S., Abouhaidar, M. G. and Campbell, J. B. Sequence analysis of putative E3 and fiber genomic regions of two strains of canine adenovirus type 1. Virology. 1991, 183, 298-305. 13. Green, C. E. Infectious canine hepatitis. In: Green, C. E. (ed.), Infectious Diseases of the Dog, and Cat. pp. 242-251. Saunders, Philadelphia, 1990. 14. Horwitz, M. S. Adenoviral diseases. In B. N. Fields, D. M. Knipe, R. M. Chanock, J. L. Melnick, B. Roizman, and R. E. Shope. eds., Virology. pp. 477-495. Raven Press, New York. 1985. 15. Hervas, J., Gomez-Villamandos, J. C., Perez, J., Carrasco, L. and Sierra, M. A. Focal mesangialsclerosing glomerulonephritis and acute-spontaneous infectious canine hepatitis: structural, immunohistochenical and subcellular studies. Vet. Immunol. Immunopathol. 1997, 57, 25-32. 16. Hu, R. L., Huang, G., Qiu, W., Zhong, Z. H., Xia, X. Z. and Yin, Z. Detection and differentiation of CAV-1 and CAV-2 by Polymerase Chain Reaction. Vet. Res. Commun. 2001, 25, 77-84. 17. Kiss, I., Matiz, K., Bajmoci, E., Rusvai, M. and Harrach, B. Infectious canine hepatitis: detection of canine adenovirus type 1 by polymerase chain reaction. Acta. Vet. Hung. 1996, 44, 253-258. 18. Kobayashi, Y., Ochiai, K. and Itakura, K. Dual infection with canine distemper virus and infectious canine hepatitis virus (canine adenovorus type 1) in a dog. J. Vet. Med. Sci. 1993, 55, 699-701. 19. Lyon, W. J. TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Iysters, and Synthetic Seawater. Appl. Environ. Microbiol. 2001, 67, 4685-4693.. 20. Ma, L., Bluyssen, H. A. and De Raeymaeker, M. Rapid determination of adenoviral vector titers by quantitative real-time PCR. J. Virol. Methods. 2001, 93, 181-188. 21. Morrison, M. D., Onions, D. E. and Nicolson, L. Complete DNA sequence of canine adenovirus type 1. J. Gen. Virol. 1997, 78, 873-878. 22. Nlesters, H. G. M., Park, D. S., Lee, Y. J., Cho, J. H., Lappalainen, M. and Chae, J. S. Hepatitis B virus DNA assay using TaqMan PCR system. Korean J. Lab. Med. 2002, 22, 246-252. 23. Nogva, H. K., Rudi, K., Naterstad, K., Holck, A. and Lillehaug, D. Applcation of 5'-nuclease PCR assay for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk. Appl. Environ. Microbiol. 2000, 66, 4266-4271. 24. Pratelli, A., Martella, V., Elia, G., Tempesta, M., Guarda, F., Capucchio, M. T., Carmichael, L. E. and Buonavoglia, C. Severe Enteric Disease in an Animal Shelter Associated with Dual Infections by Canine Adenovirus Type 1 and Canine Coronavirus. J. Vet. Med B. Infect. Dis. Vet. Public. Health 2001, 48, 385-392. 25. Pusterla, N., Chang, C. C., Chomel, B. B., Chae, J. S., Foley, J. E., DeRock, E., Kramer, V. L., Lutz, H. and Madigan, J. E. Serologic and molecular evidence of Ehrlichia spp. in coyotes in California. J. Wildl. Dis. 2000, 36, 494-499. 26. Rubarth, S. An acute virus disease with liver lesions in dogs (hepatitis contagiosa canis). A pathologicoanatomical and aetiologic investigation. Acta. Pathol. Microbiol. Scand. 1947, 24, 1-222. 27. Sasaki, N., Nakai, M., Iwamoto, I., Konishi, S. and Ikegami, T. Studies on infectious hepatitis of dogs. II. The distribution of the disease in Japan, and its immunization. Jpn. J. Vet. Sci. 1956, 18, 113-118. 28. Vishnubhatla, A. D., Fung, Y. C., Oberst, R. D., Hays, M. P., Nagaraja, T. G. and Flood, S. J. A. Rapid 5' Nuclease(TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolotica. Appl. Environ. Microbiol. 2000, 66, 4131-4135. 29. Whetstone, C. A., Draayer, H. and Collins, J. E. Characterization of canine adenovirus type 1 isolated from American black bears. Am. J. Vet. Res. 1988, 49, 778-780. 30. Willis, A. M. Canine viral infectious. Vet. Clin. North. Am. Small. Anim. Pract. 2000, 30, 1119-1133..

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