42 11, 2004, 100 ; MAHM- 40% [ 1 ] MOD [ 14 ] PCR,, [ 2-3 ] 2001 PCR,, ; [ 4 ] 5% 15% , 95%,, 30%, 80% [ 2, 5] PCR, ( Streptococcus agalactia

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1 South China Fisheries Science Vol. 11, No. 3 Jun., 2015 doi: / j. issn PCR,,,,,, (,, ) : ( Streptococcus agalactiae), cfb TaqMan PCR,, ; DNA ng L - 1, 10 ;, DNA PCR,, : ; ; PCR; TaqMan : S : A : ( 2015) Real-time quantitative PCR for detection of Streptococcus agalactiae from tilapia tissue WANG Rui, LI Liping, HUANG Ting, LIANG Wanwen, LIANG Cong, LEI Aiying, CHEN Ming ( Guangxi Key Lab. for Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fishery Sciences, Nanning , China) Abstract: By designing primers and probe based on the conserved region of cfb gene of Streptococcus agalactiae isolated from tilapia, we developed a real-time fluorescent PCR assay for detection of S. agalactiae from tilapia tissue and verified the specificity, and practicability of the assay. The standard and positive strains of S. agalactiae were positive, The minimum detectable concentration of S. agalactiae genome DNA was ng L - 1, sensitivity and all negative controls were negative. and the detection limit of the assay was less than 10 cells per reaction system. The genome DNA samples of midgut, liver, spleen and kidney which were collected from tilapia artificially infected with S. agalactiae were tested by real-time PCR and were positive. The quantities of S. agalactiae detected in per g DNA of tissue samples were cells, cells, cells and cells, respectively, and the controls were negative. The results show that the real-time quantitative PCR assay which we developed was accurate, specific and sensitive, and could detect S. agalactiae from various tilapia tissues rapidly. The method can be used for surveillance and prevention of S. agalactiae disease of tilapia. Key words: tilapia; Streptococcus agalactiae; real-time quantitative PCR; TaqMan probe : ; : : ( ) ; ( 2014GXNSFBA118083) ; ( GXYB-03 ) ; ( ) ; ( ) ; ( GXIF ) : ( ),,, raywongxx@ 163. com : ( ),,, cm990919@ 163. com

2 42 11, 2004, 100 ; MAHM- 40% [ 1 ] MOD [ 14 ] PCR,, [ 2-3 ] 2001 PCR,, ; [ 4 ] 5% 15% , 95%,, 30%, 80% [ 2, 5] PCR, ( Streptococcus agalactiae) [ 5] 1 B ( group B Streptococcus, GBS) [ 6 ], 1. 1 [ 7-8 ] g ( Oreochromis niloticus) ( ) [ 9] ATCC27956, ATCC29178 ( S. dysgalactiae) NCTC4335 ( S. parauberis), MCCCA01039 ( S. gordonii), MCCCA07812, 1. 2, 1 [ 10 ] [ 1 5] PCR, GenBank cfb ( GeneID ), ng L - 1 [ 11] PCR ( S. iniae) 1. 3 DNA ng L ng L - 1, PCR YM001,, 113 bp, ( ) YM001 ( TSB) 24 h, ( OD 600 ) PBS PCR ml BERGERON [ 12 ] PCR ml, rmin min, 180 L ; GOLDEN ( 20 Lmmol - 1 Tris, ph 8. 0; 2 mmoll - 1 Na 2- [ 13] PCR EDTA; 1. 2% Triton; 20 mgml Tab. 1 Sequences of primers and probe / primer/ probe sequence( 5 3 ) cfb-f( 53 ) forward CGGTTAATgAGGCTATTACTAGTG cfb-r( 5 3 ) reverse ATCTGTTAAGGCTTCTACACGAC cfb-p( 53 ) probe FAM-TTCATTGCGTGCCAACCCTGAGACA-Eclipse

3 3 : PCR 43 ), min DNA, C ( ) DNA, RNA min, DNA, m , 1. 5%, Q b PCR DNA Real Time PCR /TaqMan A DNA ( ) PCR,, Q Premix Ex Taq LForward Primer 1. 0 L Reverse Primer 1. 0 LTaqMan Probe 2. 0 L ROX Reference Dye II 0. 5 LDNA 4. 0 L L, 50 L; PCR 20 s; 95 3 s, s, 40 PCR cfb ( DNA ddh 2 O ) PCR ( ) PCR ATCC ATCC27956 NCTC4335 MCCCA01039 MC- ( 1) ( YM001 ATCC29178 MCCCA01039 MC- NCTC4335 CCA07812 DNA 40 CCA07812 DNA, 2), YM001 PCR DNA, 4 DNA 1. 3, 1. 6 PCR 10 9 ml - 1 DNA l0, 8 ( ), ml - 1 DNA, PCR, AB, 10 2 d A PBS YM001, , B PBS 6 h A B, Fig. 1 Amplification curve of real-time PCR specific test - 80 PBS 3 2 cm - 80,, ( ) DNA DNA 10 9 ml - 1 YM001 DNA 0. 2 g L - 1, 0. 8C= m[ log( Q) ] + b, C Y A = Q/( ) DNA ( g L - 1 ), 4 ( L) 2 100% YM001 ATCC29178 PCR DNA 3, PCR A B ATCC27956 YM001 DNA A and B represent amplification curves of standard strain ATCC27956 and test strain YM001 of S. agalactiae 2.2 PCR DNA OD 2 60 / 28 0, DNA ng L - 1, OD 2 60 /

4 PCR 2 PCR 1. ATCC27956 ( S. agalactiae) ; 2. YM001( S. agalactiae) ; 3 6. ATCC29178( S. iniae), NCTC4335 ( S. dysgalactiae), MCCCA01039 ( S. parauberis), MCCCA07812 ( S. gordonii) ; 7. ; M. DNA Fig. 2 Real-time PCR of specificity for S. agalactiae Lanes 1 7. ATCC27956( S. agalactiae), YM001( S. agalactiae), ATCC29178( S. iniae), NCTC4335 ( S. dysgalactiae), MCCCA01039 ( S. parauberis), MCCCA07812 ( S. gordonii) and blank control, respectively; M. DNA Marker( DL 2000) Fig ; M. DNA ( DL 2000) Real-time PCR of sensitivity for S. agalactiae Lanes 1 8. amplification products of the times diluted samples; M. DNA Marker( DL 2000) Fig. 5 5 PCR Real-time PCR amplification standard curves DNA PCR, 3 PCR A, B , A Fig. 3 Amplification curves of real-time PCR sensitivity test DNA The curves from left to right are amplification curves of the times diluted samples DNA 10 1 DNA lg 6, PCR ( DNA 4), 10 ml - 1 lg, DNA ( 10) PCR 3 PCR 10 DNA, PCR, ( Ct) PCR ( 5), PCR, 16S rrna A B 6 h, [ ] YM001 PBS cfb

5 3 : PCR 45 6 Fig. 6 PCR, Real time PCR detection of S. agalactiae from the tissues of tilapia DNA, CAMP 0. 2 g L - 1, [ 11, 19 ] [ 1 0], [ 1 1] cfb DNA, PCR, PCR, PCR PCR,, DNA, PCR [ ] PCR, :, [ 10 ] [ 11] PCR DNA ng L ng L DNA 10-8, ng L - 1, Ct 31 PCR 10 2,,, [ 22 ],,, DNA, PCR,, [ 12 ] [ 13 ] [ 14], 4 DNA, DNA DNA 6 h, [ 23 ] DNA DNA, [ 1] I J, LU H, ZHU J, et al. Aquatic products processing industry in China: challenges and outlook[ J]. Trends Food Sci Technol, 2009, 20( 2) : [ 2] YE X, LI J, LU M, et al. Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China [ J]. Fish Sci, 2011, 77 ( 1) : [ 3],,,. [ J]., 2013, 9 ( 6) : [ 4]. [ J]., 2002, 24( 1) : [ 5] CHEN M, LI L P, WANG R, et al. PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China[ J]. Vet Microbiol, 2012, 159 ( 3 /4) : [ 6] EVANS J J, PASNIK D J, KLESIUS P H, et al. First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin ( Tursiops truncatus) [ J]. J Wildl Dis, 2006, 42 ( 3 ) : [ 7] ZHAO Z, KONG F, MARTINEZ G, et al. Molecular serotype i- dentification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot ( mpcr / RLB) hybridization assay[ J]. FEMS Microbiol Lett, 2006, 263( 2) :

6 46 11 [ 8] HEATH P T. An update on vaccination against group B Streptococcus [ J]. Expert Rev Vaccines, 2011, 10( 5) : [ 9 ] LIU G, ZHANG W, LU C. Comparative genomics analysis of Streptococcus agalactiae reveals that isolates from cultured tilapia in China are closely related to the human strain A909[ J]. BMC Genomics, 2013, 14( 1) : 775. [ 10],,,. PCR [ J]., 2008, 47( 8) : [ J]., 2011, 41 ( 5 ) : [ 11],,,. PCR [ J]. :, 2010, 36( 4) : [ 12] BERGERON M G, KE D, MNARD C, et al. Rapid detection of group B streptococci in pregnant women at delivery [ J]. J Med, 2000, 343( 3 ) : N Engl [ 13] GOLDEN S M, STAMILIO D M, FAUX B M, et al. Evaluation of a real-time fluorescent PCR assay for rapid detection of Group B Streptococci in neonatal blood [ J]. 2004, 50 ( 1) : Diagn Microbiol Infect Dis, [ 14] MAHMMOD Y S, MWEU M M, NIELSEN S S, et al. Effect of carryover and presampling procedures on the results of real-time PCR used for diagnosis of bovine intramammary infections with Streptococcus agalactiae at routine milk recordings [ J]. Med, 2014, 113( 4) : Prev Vet [ 15],,,. PCR B [ J]., 2011, 32 ( 5) : [ 16],. PCR [ J]., 2007, 14 ( 5 ) : [ 17],,,. PCR [ 18],,. PCR [ J]., 2009, 16 ( 2 ) : 156 [ 19] HENSLER M E, QUACH D, HSIEH C J, et al. CAMP factor is not essential for systemic virulence of Group B Streptococcus[ J]. Microb Pathog, 2008, 44( 1 ) : [ 20] ARYA M, SHERGILL I S, WILLIAMSON M, et al. Basic principles of real-time quantitative PCR [ J]. 2005, 5( 2) : Expert Rev Mol Diagn, [ 21 ] BUSTIN S A, BENES V, NOLAN T, et al. Quantitative realtime RT-PCR: a perspective[ J]. J Mol Endocrinol, 2005, 34 ( 3) : [ 22 ] GUAN Q, WANG X, TENG D, et al. Construction of a standard reference plasmid for detecting GM cottonseed meal [ J]. Biochem Biotechnol, 2011, 165( 1 ) : Appl [ 23] LIESCHKE G J, TREDE N S. Fish immunology[ J]. Curr Biol, 2009, 19 ( 16 ) : R678 - R682.

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较