罗非鱼无乳链球菌环介导等温扩增(LAMP)检测技术的建立及应用
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摘要
针对罗非鱼无乳链球菌Sip基因建立了LAMP检测方法,并对野外样本进行了无乳链球菌的调查研究。结果表明,目标Sip基因可在63℃40 min内被LAMP检测到,比PCR快约2 h,其DNA检测最低浓度为3.55×10~(-5) ng/μL,灵敏度比PCR高100倍,且对参考细菌无扩增。野外样品检测结果表明,运用LAMP检测技术对GBS的检出率为81.3%,比PCR高约20.0%。应用LAMP和PCR分别检测健康罗非鱼GBS检出率4.4%和2.2%,检测池塘水样品GBS检出率分别为10.0%和6.7%。
引文
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[2]Chen M,Li L P,Wang R,et al.PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China[J].Veterinary Microbiology,2012,159(3/4):526-530.
[3]Mian G F,Godoy D T,Leal C G,et al.Aspects of the natural history and virulence of S.agalactiae infection in Nile tilapia[J].Veterinary Microbiology,2009,136(1):180-183.
[4]Evans J J,Bohnsack J F,Klesius P H,et al.Phylogenetic relationships among Streptococcus agalactiae isolated from piscine,dolphin,bovine and human sources:a dolphin and piscine lineage associated with a fish epidemic in Kuwait is also associated with human neonatal infections in Japan[J].Journal of Medical Microbiology,2008,57(11):1369-1376.
[5]Evans J J,Klesius P H,Pasnik D J.Human streptococcus agalactiae isolate in Nile tilapia (Oreochromis niloticus)[J].Emerging Infectious Diseases,2009,15(5):774-776.
[6]Bowater R O,Forbes-Faulkner J,Anderson I G,et al.Natural outbreak of Streptococcus agalactiae(GBS)infection in wild giant Queensland grouper,Epinephelus lanceolatus(Bloch),and other wild fish in northern Queensland Australia[J].Journal of Fish Diseases,2012,35(3):173-186.
[7]Jafar Q A,Sameer A Z,Salwa A M,et al.Molecular investigation of Streptococcus agalactiae isolates from environmental samples and fish specimens during a massive fish kill in Kuwait Bay[J].African Journal of Microbiology Research,2009,3(1):22-26.
[8]Pereira U P,Mian G F,Oliveira I M,et al.Genotyping of Streptococcus agalactiae strains isolated from fish,human and cattle and their virulence potential in Nile tilapia[J].Veterinary Microbiology,2010,140(1):186-192.
[9]Ye X,Li J,Lu M X,et al.Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China[J].Fisheries Science,2011,77(4):623-632.
[10]Manning S D,Springman A C,Lehotzky E,et al.Multilocus sequence types associated with neonatal group B streptococcal sepsis and meningitis in Canada[J].Journal of Clinical Microbiology,2009,47(4):1143-1148.
[11]Chaiwarith R,Jullaket W,Bunchoo M,et al.Streptococcus agalactiae in adults at Chiang Mai University Hospital:a retrospective study[J].BMC Infectious Diseases,2011,11(1):149.
[12]Lambertsen L,Ekelund K,Skovsted I C,et al.Characterisation of invasive group B streptococci from adults in Denmark 1999 to 2004[J].European Journal of Clinical Microbiology & Infectious Diseases,2010,29(9):1071-1077.
[13]Herbst K,Bujara M,Heroven A K,et al.Intrinsic thermal sensing controls proteolysis of yersinia virulence regulator RovA[J].PLOS Pathogens,2009,5(5):e1000435.
[14]Delannoy C M,Crumlish M,Fontaine M C,et al.Human Streptococcus agalactiae strains in aquatic mammals and fish[J].BMC Microbiology,2013,13(1):41.
[15]Nagamine K,Hase T,Notomi T.Accelerated reaction by loop-mediated isothermal amplification using loop primers[J].Molecular and Cellular Probes,2002,16(3):223-229.
[16]Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products[J].Nature Protocols,2008,3(5):877-882.
[17]王健胜,侯桂玲,谢永凤.国内外苜蓿品种遗传多样性RAPD分析[J].江苏农业科学,2017,45(13):35-38.
[18]李红,李超,张敏.金针菇菌株遗传多样性的RAPD分析[J].江苏农业科学,2018,46(1):19-22.
[19]Toranzo A E,Magariňos B,Romalde J L.A review of the main bacterial fish diseases in mariculture systems[J].Aquaculture,2005,246(1/4):37-61.
[20]Emaneini M,Mirsalehian A,Beigvierdi R,et al.High incidence of macrolide and tetracycline resistance among Streptococcus agalactiae strains isolated from clinical samples in Tehran[J].Maedica,2014,9(2):157-161.
[21]Poncelet-Jasserand E,Forges F,Varlet M N,et al.Reduction of the use of antimicrobial drugs following the rapid detection of Streptococcus agalactiae in the vagina at delivery by real-time PCR assay[J].BJOG,2013,120(9):1098-1109.
[22]郑磊,樊海平,吴斌,等.罗非鱼无乳链球菌LAMP快速检测方法的建立[J].福州大学学报(自然科学版),2015,43(4):572-576.
[23]Li Y W,Liu L,Huang P R,et al.Chronic streptococcosis in Nile tilapia,Oreochromis niloticus (L.),caused by Streptococcus agalactiae[J].Journal of Fish Diseases,2014,37(8):757-763.