Chinese Journal of Biochemistry and Molecular Biology PRV PCR. Detection of Pseudorabies Virus D NA by PCR. (No. 2 KM03507N),

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1 ISSN CN 23870ΠQ Chinese Journal of Biochemistry and Molecular Biology 7 (4) :59523 PCR 3 ( 50640) (PRV) gb A PRV PCR Fb Bartha BJ GD V2F4 S S3 SR Buk Shope Norden Mink HB F8 F9 F2 28 bp Vero FMDV SVDV HCV PRRSV J EV PPV PRV PCR PRV ELISA PCR 3 2 PCR ;PCR 2 ; 2 PCR ;x 2 PCR 2 PRV A 58 pg %(50Π9) 7 %(22Π3) PCR PCR S Detection of Pseudorabies Virus D NA by PCR LOU Gao2ming DU Wei2xian LIAO Xiao2Ping XIE Ming2quan ( The Guangdong Key Laboratory of Veterinary Biotechnology Guangzhou China) Abstract One pair of primers that amplified the gb gene of pseudorabies virus (PRV) was designed and syn2 thesized PCR technique detecting the of PRV was established after selecting the best reaction conditions This technique was applied to specifically amplify the 28 bp fragment of the PRV strains including Fa Fb Bartha BJ GD V2F4 S S3 SR Buk Shope Norden Mink HB F8 F9 and F2 in cultured samples The negative results were achieved from Vero cells swine vesicular disease virus hog cholera virus Japanese encephalitis virus porcine reproductive and respiratory syndrome virus porcine parvovirus foot2and2mouth dis2 ease virus F29 strain O3I3 strain T509 strain and O MF249 strain The results of sequencing showed that the PCR method was of specificity The sensitivity of PCR reached 5 8 pg of PRV Fa strain The tissue samples obtained during 994 and 2000 were detected and the results showed that the sensitivity of PCR was more sensitive than virus isolation and the Sandwich ELISA The PCR was applied to detect 9 tissue samples from 3 pig farms obtained from Guangdong Fujian Hainan Provinces during 999 and samples(26 2 %) were positive and 22 pig farms(7 %) were positive The results showed that established PRV PCR tech2 nique provided a more sensitive specific and reliable method for diagnosis and epizootic study of the pseudora2 : : (No 962C ) (No 2 KM03507N) (No ) (No 99057) 3 : (9656) Tel : (020) Fax : (020) E2mail net Received :November ;Accepted :January Supported by the Ministry of Science and Technology(No 962C ) Guangdong Provincial Department of Science and Technology(No 2KM03507N) National Natural Science Foundation of China (No ) ; Guangdong Provincial Natural Science Foundation of China (No 99057) 3 Correspionding author Tel : (020) Fax : (020) E2mail net

2 520 7 bies Key words pseudorabies virus detection PCR (pseudorabies PR) (J EV) ( PPV) ( FMDV) F29 (pseudorabies virus PRV) O33 T509 O MF249 ( ) ; (Vero ) DMEM GIBCO ; ( ) ; 3 Taq Sangon ( A20) Boehringer Mannheim GmbH ( PR ) dntps ( () 79A) 00 bp Ladder kb Ladder MBI Biolabs 4 PRV Vero 0 % ( 00 IUΠml 00gΠml) DEME (75 % NaHCO 3 ph 7274) 2 % DMEM 75 % 37 h % [3 ] 5 PCR PCR 5 PRV Fa PRV Fa PRV 75 cm [48 ] PCR PRV l (00 molπl Tris2HCl ph molπ L EDTA 0 25 % Triton X molπl NaCl 2 5 % PRV A ( Fa) ; B 0 min rπmin 5 min (Fb) Bartha Π2 Π 3 ;BJ l RNA (0 mgπml) 37 5 min 5 ; GD V2F4 molπl NaCl 0 molπl 25 ; ( S) ( S3) 0 min (SR) Buk Shope Norden Mink rπmin 30 min 70 % 4 ; ( HB) 000 rπmin 5 min 50 l TE ( ;F8 F9 mmolπl Tris2HCl 0 mmolπl EDTA ph 8 0) - F2 TK ge 20 Sarkosyl) K(0 mgπml) 25 l 37 5 min 5 ml 400 l ; (SVDV) ( HCV) 52 PRV ( PRRSV)

3 4 :PCR 52 PBS (ph 7 2) l l min 200 l rπmin 5 min Π 2 PCR ELISA min rπmin 0 min 70 % rπmin 5 min 20 l TE [9 ] PRV ( P ) : 5 2CATCAC2 CACGGGCTCGGCGGAGTTT23 gb ; ( P2) : 5 2TACGTGCAGAACTC2 CATGCGCGTGC23 gb ; 28 bp PCR 7 PCR [0 ] 2 PCR PCR 8 PCR : 50 l 0 05 l 2 % ( 5 l 25molΠL dntps l 0molΠL ( P P2 05gΠml ) TAE ) l 25molΠL MgCl 2 3 l 0 l (0 04 molπl Tris molπl EDTA ph80) 5 l ( 0 %) 245 l 20 V 3060 min PCR 95 5 min Taq bp Ladder kb l (2 5 U) 50 l Ladder 9 PCR PRV PCR PRV Fb Bartha BJ GD V2F4 S S3 SR Buk Shope Norden Mink HB F8 F9 F2 FMDVF29 O3I3 T509 O MF249 SVDV HCV PRRSV J EV PPV F2 ( Fig ) ; Vero Vero 0 PCR PRV PRV Fa MF249 ( Fig 2) 0 l PRV PCR ( ) PCR PCR 2 23 PCR PRV ( 00 IUΠ PRV Fa (58 ngπml) ml 00gΠml) rπmin 0 min 02m PCR ; 24 PCR 2 Vero d 34 5 PRV PCR ELISA [ ] PCR 3 PCR PCR PCR :93 min 65 min 72 5 min 30 ; 72 5 min 22 PCR PRV PRV Fa Fb Bar2 tha BJ GD V2F4 S S3 SR Buk Shope Norden Mink HB F8 F9 SVDVHCV J EV PRRSV PPV FMDV F29 O3I3 T509 O 0 PCR 58 pg PRV

4 Table Results of samples tested by PCR and virus isolation PRV PCR + - Virus isolation Table 2 Results of samples tested by PCR and the Sandwich ELISA PCR + - Sandwich ELISA Fig Results of different PRV strains detected by PCR A :00 bp ladder ; B :Fa strain ; C:F9 strain ; D :Buk strain ; E:BJ strain ; F :V2F4 strain ; G:S strain ; H: GD strain ; I :F2 strain 25 PCR ELISA PCR ELISA 2 Table 2PCR 463 % (3Π67) ELISA 343 % (23Π67) x 2 ( P 005) 2 PCR ELISA 26 PCR PCR % (50Π9) ; 22 7 %(22Π3) Fig 3 Fig 2 Results of different viruses detected by PCR A :00 bp ladder ; B :Fb strain ; C:J EV ; D :HCV ; E:PRRSV ; F :SVDV ; G:FMDV O3I3 strain ; H:PPV ; I : kb Ladder ; PCR Table PCR 7083 % (7Π24) 297 % (7Π24) x 2 Fig 3 Results of different tissue samples by PCR ( P 00) 2 A :00 bp ladder ; B :F8 strain ; C Negative control ; PCR D :Brain tissue ; E:Tonsil tissue ; F :Spleen tissue ; G:Lung tissue ; H:Liver tissue ; I : kb Ladder

5 4 :PCR PRV ( ) PRV ) ( ) ( References) PCR PRV PCR : SVDVHCV J EV PRRSV PPV FMDV F29 O3I3 T509 O 998 : : ( Yin MF249 Vero ; PRV 00 % 37 d PCR 6 h ELISA PCR 2 2 PCR ; PCR 2 ; 2 PCR 20 d PCR PCR ELISA : ; ; PRV PRV 6 PCR (Lou Gao2ming Guo Wan2zhu Xu Lan2fang ed Breeding Technology Prevention and Treatment in Intensive Pig In2 dustry Changchun : Jilin Provincial Science and Technology Press) ZhenLiu Jing2hua ed Virology in Animal 2nd ed Beijing : Science Press) 997 : (Lou Gao2ming Du Wei2xian Advances on research of serological methods of pseudorabies Animal Sci Vet Med) () :02 4 Volz D M Lager K M Mengelin W L Latency of a thymidine kinase2 negative pseudorabies vaccine virus detected by the polymerase chain re2 action Arch Virol : Harding MJ Prud homme I Rola J Specificity and nucleotyping studies of a gp50 - based polymerase chain reaction assay for detection of pseud2 orabies virus Can J Vet Res : Maes R KBeisel C E Spatz SJ Thacker B J Polymerase chain reaction amplification of pseudorabies virus from acutely and latently infect2 ed cells Vet Microbiol : Thiery R Pannetier C Rziha HJ Jestin A A fluorescence2based quanti2 tative PCR method for investigation of pseudorabies virus latency J Virol Methods : Cheung A K Detection of the large latency transcript of pseudorabies virus by RNA2PCR and its potential in diagnosis J Vet Diag Invest : Robbins A KDorney D J Wathen M W Whealy M E Gold C Watson R J Holland L E Weeds D Levine M Glorioso J C Enquist L W The pseudorabies virus g gene is closely related to the gb glycoprotein gene of herpes simplex virus J Virol : Sambrook J Fritsch E F Maniatis T Molecular Cloning : A Laboratory Manual 2nd ed New York :Cold Spring Harbor Laboratory Press 989 : 20(4) : ELISA (Lou Gao2ming Chen Zhi2rong Guo Wan2zhu Wang Qin Wang Ming2 shu Yan Qi2gui Zou Xiao2huan Fei En2ge Detection of pseudorabies virus with the Sandwich ELISA Chin J Animal Poult Infect Dis) 998