Development and Preliminary Application of Loop-mediated Isothermal Amplification （LAMP） Method for Rapid Detection of Streptococcus iniae

doi:10.14088/j.cnki.issn0439-8114.2018.15.025

Abstract

Abstract: To develop a rapid and accurate method for detecting Streptococcus iniae and limit the losses because of this disease, a loop-mediated isothermal amplification （LAMP） assay was established with four specific primers designed according to the Sim gene of Streptococcus iniae in GenBank, and totally 739 samples were collected and detected by LAMP. The results showed that the LAMP had a high sensitivity for S. iniae with detection limits of 5.32 copies/μL S. iniae DNA. The LAMP method had a strong specificity, which can be specifically amplified S. iniae, and no amplifications were observed in the 8 reference strains. The detection results of 739 samples showed that the average detection rate of S. iniae in the samples from 8 species of cultured fish was 17.67%, while 13.33% and 16.67% were from pond water and seawater, respectively. The bacteria were found mainly in tilapia brain tissues, and the average positive detection rate was 27.04%, while in other fishes, the average positive detection rate was 24.14%. Higher S. iniae positive carrying rate during Jun to Sep, 2014 was found comparing to Jan to May and Oct to Dec, 2014, with the detection rate reached to 65.00%.

Key words: Streptococcus iniae, streptococcosis, loop-mediated isothermal amplification （LAMP）, tilapia

CLC Number:

S852.61

ZHANG Yong-de, FENG Shi-wen, LI Jun, PENG Hao, LIANG Wan-wen, YU Yan-ling, LI Ming, YANG Xue-ming, ZHU Jia-jie, LUO Hong-lin, CHEN Fu-yan, LEI Ai-ying. Development and Preliminary Application of Loop-mediated Isothermal Amplification （LAMP） Method for Rapid Detection of Streptococcus iniae[J]. HUBEI AGRICULTURAL SCIENCES, 2018, 57(15): 99-104.

References

[1] SUANYUK N,SUKKASAME N,TANMARK N,et al. Streptococcus iniae infection in cultured Asian sea bass(Lates calcarifer) and red tilapia(Oreochromis sp.) in southern Thailand[J].Songklanakarin Journal of Science & Technology,2010,32(4):341-348.
[2] DUREMDEZ R,AL-MARZOUK A,QASEM J A,et al.Isolation of Streptococcus agalactiae from cultured silver pomfret,Pampus argenteus(Euphrasen),in Kuwait[J].Journal of fish Diseases, 2004,27(5):307-310.
[3] ANSHARY H,KURNIAWAN R A,SRIWULAN S,et al.Isolation and molecular identification of the etiological agents of streptococcosis in Nile tilapia(Oreochromis niloticus) cultured in net cages in Lake Sentani,Papua,Indonesia[J].Springer Plus,2014,3(1):627.
[4] ABDELSALAM M,EISSA A E,CHEN S C.Genetic diversity of geographically distinct Streptococcus dysgalactiae isolates from fish[J].Journal of Advanced Research,2015,6(2):233-238.
[5] ALI A,HASSAN D,SALEHA A A,et al.Streptococcus agalactiae the etiological agent of mass mortality in farmed red tilapia (Oreochromis sp.)[J].Journal of Animal and Veterinary Advances,2010,9(20):2640-2646.
[6] AGNEW W,BARNES A C.Streptococcus iniae:An aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination[J].Veterinary microbiology, 2007,122(1):1-15.
[7] 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.
[8] LOW D E,LIU E,FULLER J,et al.Streptococcus iniae: An emerging pathogen in the aquaculture industry[M].Washington. DC:ASM Press;1999.
[9] BAIANO J C F,BARNES A C. Towards control of Streptococcus iniae[J].Emerging Infectious Diseases,2009,15(12):1891-1896.
[10] DODSON S V,MAURER J J,SHOTTS E B.Biochemical and molecular typing of Streptococcus iniae isolated from fish and human cases[J]. Journal of Fish Diseases,1999,22(5):331-336.
[11] GOH S H, DRIEDGER D, GILLETT S, et al.Streptococcus iniae,a human and animal pathogen: Specific identification by the chaperonin 60 gene identification method[J]. Journal of Clinical Microbiology,1998,36(7):2164-2166.
[12] MATA A I,BLANCO M M,DOMNGUEZ L,et al.Development of a PCR assay for Streptococcus iniae based on the lactate oxidase (lctO) gene with potential diagnostic value[J]. Veterinary Microbiology,2004,101(2):109-116.
[13] TORANZO A E,MAGARINOS B,ROMALDE J L.A review of the main bacterial fish diseases in mariculture systems[J]. Aquaculture,2005,246(1):37-61.
[14] 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.
[15] CAI S H,WANG B,LU Y S,et al.Development of Loop-mediated isothermal amplification method for rapid detection of Streptococcus iniae, the causative agent of streptococcicosis in fish[J]. Journal of Basic Microbiology,2012,52(2):116-122.
[16] HAN H J,JUNG S J,OH M J,et al.Rapid and sensitive detection of Streptococcus iniae by loop-mediated isothermal amplification(LAMP)[J].Journal of Fish Diseases,2011,34(5): 395-398.
[17] SUEBSING R,KAMPEERA J,TOOKDEE B,et al.Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia[J].Letters in Applied Microbiology,2013,57(4):317-324.
[18] BAIANO J C F,TUMBOL R A,UMAPATHY A,et al. Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae[J]. BMC Microbiology,2008, 8(1):1-16.
[19] BUCHANAN J T,STANNARD J A,LAUTH X,et al.Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development[J].Infection and Immunity,2005, 73(10):6935-6944.
[20] MILLER J D,NEELY M N.Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae[J]. Infection and Immunity,2005,73(2): 921-934.
[21] BARNES A C,YOUNG F M,HORNE M T,et al.Streptococcus iniae: Serological differences, presence of capsule and resistance to immune serum killing[J]. Diseases of Aquatic Organisms,2003,53(3):241-247.
[22] LOCKE J B,COLVIN K M,DATTA A K,et al.Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish[J]. Journal of Bacteriology,2007,189(4): 1279-1287.
[23] NGUYEN H T,KANAI K,YOSHIKOSHI K.Ecological investigation of Streptococcus iniae in cultured Japanese flounder(Paralichthys olivaceus) using selective isolation procedures[J]. Aquaculture,2002,205(1):7-17.