Microsoft Word - 09 黄锦炉.doc

Size: px

Start display at page:

Download "Microsoft Word - 09 黄锦炉.doc"

梁透太叔
7 years ago
Views:

1 43 2 Vol.43, No OCEANOLOGIA ET LIMNOLOGIA SINICA Mar., 2012 (Streptococcus agalactiae) PCR * 1 1, ( ; ; ) GenBank sip cpse, cpsl,, PCR, PCR 40 22, PCR, DNA, 7 DNA, DNA 0.32ng/μl, Mg mmol/L, 100min 40 DNA 100%, 22 DNA 72.7%,, PCR, Q7 (Streptococcus agalactiae), B (group B streptococci, GBS), (Schuchat, 1998; Trigo et al, 2008;, 2008; 璟, 2010),,, (Amal et al, 2011), ( 璟, 2010), ; (Rosa et al, 1995; Yancey et al, 1993),, ; PCR (Mawn et al, 1993; Ahmet et al, 1999),,,,, PCR,, (, 2003), (Elnifro et al, 2000), (Surface Immunogenic *, IRT0848 ;, ,, hgl25@163.com :,,, kywang@sicau.edu.cn : , :

2 2 : (Streptococcus agalactiae) PCR 255 Protein) sip (Rioux et al, 2001) (Galactosyltransferase) cpse (Chaffin et al, 2005;, 2011), (Polysaccharide Biosynthesis Protein) cpsl (Imperi et al, 2010) bp,, PCR, PCR, sip cpse cpsl, PCR, (Streptococcus agalactiae) (ATCC51487) (S. iniae) (ATCC29178) (Edwardsiella ictaluri) (Aeromonas hydrophila) (A. veronii) (Stenotrophomonas maltophilia) (Yersinia ruckeri) (A. sobria) 1.2 MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0(TaKaRa, DV810A); DNA Marker PCRmix Goldgreen( ); (Sigma) 1.3 Imperi (2010) cpsl S. agalactiae,,, S. agalactiae sip cpse, sip ( DQ650634) cpse ( AB017355) DNA, 30 24h, DNA (TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0) DNA 1.5 PCR cpse cpsl sip PCR : Master Mix 12.5μl, (10mmol/L) 0.5μl, DNA (10ng/μl) 1μl, ddh 2 O 25μl; : 94 5min, 94 30sec, 55 35sec, 72 1min, 72 10min, 30 PCR, 5μl, 100V, 1% 30min PCR 1.6, cpse 10μmol/L cpsl 2μmol/L sip 2μmol/L PCR,, 2μmol/L cpse (0.6μl 0.6μl) (0.3μl 0.3μl) (0.1μl 0.1μl), A 1 A 2 A 3 ; 2μmol/L cpsl (0.8μl 0.8μl) (0.4μl 0.4μl) (0.2μl 0.2μl), B 1 B 2 B 3 ; 2μmol/L sip (0.8μl 0.8μl) (0.4μl 0.4μl ) (0.2μl 0.2μl), C 1 C 2 C 3 A x B y C z (x = 1, 2, 3; y = 1, 2, 3; z = 1, 2, 3), 2 表 1 三组引物的信息 Tab.1 The information of the three primers (5 3 ) (bp) (bp) cpse ATGAAAATTTGTCTGGTTGG cpse TTAAAAAATTCCTCCTAAATT cpsl CAATCCTAAGTATTTTCGGTTCATT cpsl TAGGAACATGTTCATTAACATAGC sip ATGGAAATGAATAAAAAGGTAC sip TTAGTTAAAGGATACGTGAACGTGG

3 Tab.2 表 2 三对引物组合表 The combinative table of the three primers 2μmol/L cpse 2μmol/L cpsl(μl) (μl) B 1 : (0.8, 0.8) B 2 : (0.4, 0.4) B 3 : (0.2, 0.2) 10μmol/L sip (μl) A 1 : (0.6, 0.6) A 1 B 1 C 1 A 1 B 2 C 1 A 1 B 3 C 1 C 1 : (0.8, 0.8) A 1 B 1 C 2 A 1 B 2 C 2 A 1 B 3 C 2 C 2 : (0.4, 0.4) A 1 B 1 C 3 A 1 B 2 C 3 A 1 B 3 C 3 C 3 : (0.2, 0.2) A 2 : (0.3, 0.3) A 2 B 1 C 1 A 2 B 2 C 1 A 2 B 3 C 1 C 1 : (0.8, 0.8) A 2 B 1 C 2 A 2 B 2 C 2 A 2 B 3 C 2 C 2 : (0.4, 0.4) A 2 B 1 C 3 A 2 B 2 C 3 A 2 B 3 C 3 C 3 : (0.2, 0.2) A 3 : (0.1, 0.1) A 3 B 1 C 1 A 3 B 2 C 1 A 3 B 3 C 1 C 1 : (0.8, 0.8) A 3 B 1 C 2 A 3 B 2 C 2 A 3 B 3 C 2 C 2 : (0.4, 0.4) A 3 B 1 C 3 A 3 B 2 C 3 A 3 B 3 C 3 C 3 : (0.2, 0.2) PCR PCR 25μl, 3 PCR, 5μl, 1μl 6 Loading Buffer, 100V, 1% 30min cpse cpsl sip,, PCR 表 3 反应体系试剂组成 Tab.3 The content of the reagent in the reaction system (μl) 10 Buffer mmol/L MgCl dntp mmol/L DNA 1.0 5U/μl Taq 0.5 A x B y C z 4 x = 1, 2, 3; y = 1, 2, 3; z = 1, 2, 3 ddh 2 O (ATCC51487) (ATCC29178) DNA, 1.6, 3 PCR, 100V, 1% 30min, (ATCC51487) DNA, DNA 1.8 SmartSpec plus, (ATCC51487) DNA, DNA 80ng/μl, DNA, DNA, 1.7 PCR, PCR DNA 1.9 Mg , Mg 2+, Mg 2+ ( 4) : 25mmol/L MgCl 2, 0μl 0.5μl 1.0μl 1.5μl 2.0μl 2.5μl, DNA, PCR,, Mg 2+ Tab.4 表 4 反应体系试剂组成 The content of the reagent in the reaction system (μl) 10 Buffer mmol/L MgCl 2 V V = 0, 0.5, 1.0, 1.5, 2.0, 2.5μl dntp mmol/L DNA 1.0 5U/μl Taq 0.5 A x B y C z 4 x = 1, 2, 3; y = 1, 2, 3; z = 1, 2, 3 ddh 2 O

4 2 : (Streptococcus agalactiae) PCR 257 DNA ( 1.4), PCR DNA 3, ATCC51487 DNA DNA 1.11 PCR, 40 22,, DNA Takara Universal Genomic DNA Extraction Kit Ver ( 1A) 2.2 cpse cpsl sip A x B y C z 27, PCR, A 2 B 2 C 1 PCR ( 1B), cpse (2μmol/L) cpsl (2μmol/L) sip (10μmol/L) (0.3μl 0.3μl) (0.4μl 0.4μl) (0.8μl 0.8μl), , DNA,,,,, PCR, 2μmol/L cpse (0.3μl 0.3μl) + 2μmol/L cpsl (0.4μl 0.4μl) + 10μmol/L sip (0.8μl 0.8μl), DNA ( 9) (ATCC51487)DNA ( 10),, (ATCC29178) DNA, ( 2A) 2.4 DNA 1 PCR A 2 B 2 C 1 Fig.1 The amplification with the single primer, and the primers compose A 2 B 2 C 1 in different temperatures A. PCR, sip cpsl cpse B. A 2 B 2 C 1, 1: 49 ; 2: 51 ; 3: 53 ; 4: 55 ; 5: 57 ; 6: 59 M: DL2000 Marker bp, sip ; bp, cpsl ; bp, cpse, sip cpsl cpse sip cpsl cpse 100% 100% 100% 2 PCR DNA Fig.2 The specificity result of triple PCR assay, and the sensitivity result of the S. agalactiae DNA template A. PCR, 1: (ATCC29178); 2: ; 3: ; 4: ; 5: ; 6: ; 7: ; 8: ; 9: ; 10; (ATCC51487) B. DNA, 1: DNA 10 1 ng/μl; 2: DNA 10 2 ng/μl; 3: DNA 10 3 ng/μl; 4: DNA 10 4 ng/μl; 5: DNA 10 5 ng/μl M: DL2000 Marker

5 PCR, DNA ng/μl, ( 1); DNA ng/μl,, sip cpse ; DNA ng/μl, sip cpse, cpsl ; DNA ng/μl ng/μl, sip cpsl cpse 2B 2.5 Mg 2+ A 2 B 2 C 1 DNA 8ng/μl, Mg 2+ Mg 2+, ( 1); Mg mmol/L, sip cpse, cpsl ( 2); Mg 2+ 25mmol/L 50mmol/L 62.5mmol/L,, ; Mg mmol/L,, Mg 2+, PCR Mg mmol/L, DNA 3A μl, A 2 B 2 C 1 Mg mmol/L dntp 2.5mmol/L Taq 2.5U/μl, 5 DNA DNA ( 1 5),, 3B 3 Mg 2+ Fig.3 The optimization result of the Mg 2+ concentration, and the repeatability result of the detection A. Mg 2+, 1: 0; 2: 12.5mmol/L; 3: 25mmol/L; 4: 37.5mmol/L; 5: 50mmol/L; 5: 62.5mmol/L B., 1 5: DNA DNA ; 6: DNA DNA M: DL2000 Marker , PCR 100%, 22, PCR 16,, ( 5) PCR,, (Hagay et al, 1993); 表 5 样品检测结果 Tab.5 The detection result of the samples / (%) 40/ PCR 40/ / PCR 40/ / PCR 16/ / PCR 16/

6 2 : (Streptococcus agalactiae) PCR 259, (Gil et al, 1999; Teixeira et al, 1992; Wang et al, 1988);, (Philipson et al, 1995),,, Greisen (1994) 16S rrna,,, ; Ahmet (1999), PCR,, β-, γ-, γ-,,,,, cpsl sip cpse, PCR,, PCR γ-, γ- PCR 3.2 PCR, ;,, cpse cpsl sip,, PCR, cpse cpsl sip, ; PCR, cpse cpsl sip (ATCC51487) DNA, (ATCC29178) 7 DNA,,, 7, 25μl DNA <8ng, DNA <0.032ng/μl, PCR, sip cpse, DNA 0.32ng/μl, PCR, PCR 0.32ng/μl, 100min,,, 3.3 PCR PCR, Mg 2+ Taq dntp, PCR (, 2008),, (Henegariu et al, 1997), 25μl 10 Buffer 2.5μl 25mmol/L MgCl 2 1.5μl 2.5mmol/L dntp 1.0μl DNA 1.0μl 5U/μl Taq 0.5μl, sip cpsl cpse,,, Sip, cpsl cpse sip cpsl cpse,, PCR sip cpsl cpse 4 27, A 2 B 2 C 1,,, sip cpsl cpse, PCR Taq ( Taq polymerase 5U/μl, 10 Taq PCR Buffer) 25mmol/L MgCl 2 2.5mmol/L dntp mix, PCR, DNA, DNA

7 (Henegariu et al, 1997) PCR Mg 2+, Mg 2+,, Mg mmol/L, Mg 2+ PCR ; Mg mmol/L PCR, dntp 200μmol/L Taq 0.5U, PCR Mg mmol/L, dntp Taq Mg 2+, Chamberlain (1988) 3.4 PCR PCR, PCR,,,, DNA ;, PCR,, (, 2011) PCR, (Chamberlain et al, 1992),, PCR PCR Taq, PCR 70s, 51 59, A2B2C1, ; 51 59, , 70s, A 2 B 2 C , 55,,, cpse., 35(5): ,,, , 32(5): ,,, PCR., 33(11): ,,, DNA PCR., 27(4): 璟,,, (Oreochromis niloticus)., 41(4): ,,, PCR., 25(1): Ahmet Z, Stanier P, Harvey D et al, New PCR primers for the sensitive detection and specific identification of Group B b-hemolytic streptococci in cerebrospinal fluid. Molecular and Cellular Probes, 13(5): Amal M N A, Zamri-Saad M, Streptococcosis in Tilapia (Oreochromis niloticus): A Review. Pertanika Journal of Tropical Agricultural Science, 32(2): Chaffin D O, Mentele L M, Rubens C E, Sialylation of group B streptococcal capsular polysaccharide is mediated by cpsk and is required for optimal capsule polymerization and expression. Journal Of Bacteriology, 187(13): Chamberlain J S, Gibbs R A, Ranier J E et al, Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research, 16(23): Chamberlain J S, Gibbs R A, Ranier J E et al, Detection of gene deletions using multiplex polymerase chain reactions. Methods in Molecular Biology, 9(12): Elnifro E M, Ashshi A M, Cooper R J et al, Multiplex PCR: optimization and application in diagnostic virology. Clinical Microbiology Reviews, 13(4): Gil E G, Rodriguez M C, Bartolome R et al, Evaluation of the Granada agar plate for detection of vaginal and rectal group B streptococci in pregnant women. Journal of clinical Microbiology, 37(8): Greisen K, Loeffelholz M, Purohit A et al, PCR primers and probes for the 16S rrna gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. Journal of clinical Microbiology, 32(2): Hagay Z J, Miskin A, Goldchmit R et al, Evaluation of two rapid tests for detection of maternal endocervical group B streptococcus: enzyme-linked immunosorbent assay and Gram stain. Obstet Gynecol, 82(1): Henegariu O, Heerema N A, Dlouhy S R et al, Multiplex PCR: critical parameters and step-by-step protocol. Bioteehnique, 23(3): Imperi M, Pataracchia M, Alfarone G et al, A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae. Journal of Microbiological Methods, 80(2):

8 2 : (Streptococcus agalactiae) PCR 261 Mawn J A, Simpson A J, Heard A J, Detection of the C protein gene among group B streptococci using PCR. Journal of Clinical Pathology, 46(7): Philipson E H, Palermino D A, Robinson A, Enhanced antenatal detection of group B streptococcus colonization. Obstet Gynecol, 85(3): Rioux S, Martin D, Ackermann H W et al, Localization of surface immunogenic protein on Group B Streptococcus. Infection and Immunity, 69(8): Rosa C, Clark P, Duff P, Performance of a new DNA probe for the detection of group B streptococcal colonization of the genital tract. Obstet Gynecol, 86(4): Schuchat A, Epidemiology of group B streptococcal disease in the United States: shifting paradigms. Clinical Microbiology Reviews, 11(3): Teixeira L A, Figueiredo A M S, Benchetrit L C, Liquid medium for rapid presumptive identification of group B streptococci. Journal of clinical Microbiology, 30(2): Trigo G, Ferreira P, Ribeiro N et al, Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis. Can J Microbiol, 54(11): Wang E E L, Hammerberg O, Lyn P et al, Rapid detection of group B streptococcal carriage in parturient women using a modified starch serum medium. Clinical and Investigative Medicine, 11(1): Yancey M K, Clark P, Armer T et al, Use of a DNA-probe for the rapid detection of group B streptococci in obstetric patients. Obstet Gynecol, 81(4): THE DEVELOPMENT AND APPLICATION OF A TRIPLE PCR METHOD FOR RAPID DETECTION OF STREPTOCOCCUS AGALACTIAE HUANG Jin-Lu 1, WANG Kai-Yu 1, 2, XIAO Dan 3, LIU Bei-Bei 1, WANG Jun 1, HUANG Ling-Yuan 1, FU Xi 1, WANG Hao-Cheng 1 (1. Fish Disease Research Center of Sichuan Agricultural University, Ya an, ; 2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya an, ; 3. Tongwei Co. Ltd., Chengdu, ) Abstract A pair of specific primers were designed based on the two well conserved genes of sip and cpse of Streptococcus agalactiae published on the GenBank, respectively. The primer of cpsl gene well conserved in S. agalactiae was chosen to be the positive control. After the optimization of the reaction conditions and reaction system parameters, the triple PCR method for S. agalactiae detection was developed. The method was evaluated by examining samples of forty tilapia with artificial infection and twenty two suspected fish collected clinically. Results showed that the triple PCR method was well specific. The detection result of S. agalactiae DNA showed three bands, and those of other seven strains showed no bands. The minimum detection concentration of the S. agalactiae DNA was 0.32ng/μl, the optimization value of Mg 2+ concentration was 37.5mmol/L, and it took about 100min to perform the examination. The positive rates of forty liver DNA samples and forty kidney DNA samples from the tilapia with artificial infection were 100%, respectively, and those of the twenty two liver DNA samples and twenty two kidney DNA samples from the suspected fish were 72.7%, respectively. Detection results of the two groups of samples were consistent with the results using general bacterial identification method. Key words Streptococcus agalactiae, Triple PCR, Detection method

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus