A real-time fluorescence loop-mediated isothermal amplification method for the detection of Anisakis simplex
-
摘要: 为了快速鉴定简单异尖线虫,本研究建立了一套检测简单异尖线虫DNA的恒温实时荧光环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法,根据LAMP方法原理,针对简单异尖线虫ITS2区域设计引物特异性识别靶标基因。进行了特异性、灵敏度、重复性和实际样品的测试,并与传统的PCR方法进行比较。结果表明,该方法能够特异性扩增简单异尖线虫DNA,对含有简单异尖线虫ITS2目的基因片段的质粒DNA检测限为1 fg/μL,灵敏度比传统的PCR方法高100倍,重复性良好,对实际样品进行检测,与传统的PCR测序方法结果相符。本研究建立的恒温实时荧光快速检测方法适用于特异性检测简单异尖线虫。Abstract: We established a real- time fluorescence loop- mediated isothermal amplification( LAMP) approach for the sensitive,rapid and reliable detection of Anisakis simplex.The method was based on LAMP reaction.Three sets of the specificity and sensitivity of primers were designed from ITS2 r DNA. The specificity and sensitivity were tested.Specific amplification products were obtained with A.simplex,while no amplification products were detected with DNA of related parasites,demonstrating the specificity of the assay.The capable detection of plasmid DNA in the method was 1 fg / μL.The real- time fluorescence LAMP assay was proved to be 100 times more sensitive than a conventional polymerase chain reaction for detection of A. simplex. Samples testing by the real- time fluorescence LAMP method and PCR method showed that,the result of the LAMP method was the same with PCR method.The established real- time fluorescence LAMP method was suitable for specifically detecting A.simplex.
-
[1] Judy A Sakanari,James H Mckerrow.Anisakiasis[J].Clinical Microbiology Reviews,1989,2(3):278-284.
[2] Valle J,Lopera E,Sanchez ME,et al.Spontaneous splenic rupture and Anisakis appendicitis presenting as abdominal pain:a case report[J].J Med Case Rep,2012,6:114.
[3] Arizono N,Yamada M,Tegoshi T,et al.Anisakis simplex sensustricto and Anisakis pegreffii:Biological Characteristics and Pathogenetic Potential in Human Anisakiasis[J].Foodborne Pathogens and Disease,2012,9(6):517-521.
[4] Mattiucci S,Fazii P,De Rosa A,et al.Anisakiasis and Gastroallergic Reactions Associated with Anisakis pegreffii Infection,Italy[J].Emerging Infectious Diseases,2013,19(3):496-499.
[5] Quiazon KMA,Zenke K,Yoshinaga T.Molecular characterization and comparison of four Anisakis allergens between Anisakis simplex sensu stricto and Anisakis pegreffii from Japan[J].Molecular and Biochemical Parasitology,2013,190(1):23-26.
[6] Nieuwenhuizen NE,Lopata AL.Allergic Reactions to Anisakis Found in Fish[J].Current Allergy and Asthma Reports,2014,14(8):455.
[7] Baird FJ,Gasser RB,abbar A,et al.Foodborne anisakiasis and allergy[J].Molecular and Cellular Probes,2014,28(4):167-174.
[8] Chai J,Darwin Murrell K,Lymbery AJ.Fish-borne parasitic zoonoses:Status and issues[J].International Journal for Parasitology,2005,35(11-12):1233-1254.
[9] 张望,陈秀开,李正高,等.异尖线虫病研究进展[J].动物医学进展,2014,7(35):93-97. [10] Bernardi C,Gustinelli A,Fioravanti ML,et al.Prevalence and mean intensity of Anisakis simplex(sensu stricto)in European sea bass(Dicentrarchus labrax)from Northeast Atlantic Ocean[J].International Journal of Food Microbiology,2011,148(1):55-59.
[11] Cipriani P,Smaldone G,Acerra V,et al.Genetic identification and distribution of the parasitic larvae of Anisakis pegreffii and Anisakis simplex(s.s.)in European hake Merluccius merluccius from the Tyrrhenian Sea and Spanish Atlantic coast:Implications for food safety[J].International Journal of Food Microbiology,2015,198:1-8.
[12] 中国科学院水生生物研究所、全国水产技术推广总站.SC/T 7210-2011,鱼类简单异尖线虫幼虫检测方法[S].北京:中国农业出版社,2011. [13] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[14] 陈信忠,郑洋妹,龚艳清.环介导等温扩增(LAMP)技术鉴定简单异尖线虫[A].动物检疫学分会2010年年会论文集[C].2010. [15] Zhu XQ,Gasser RB,Podolska M,et al.Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal DNA sequences[J].International Journal for Parasitology,1998,28(12):1911-1921.
[16] Qin Y,Zhao Y,Ren Y,et al.Anisakiasis in China:The First Clinical Case Report[J].Foodborne Pathogens and Disease,2013,10(5):472-474.
[17] Fu S,Qu G,Guo S,et al.Applications of Loop-Mediated Isothermal DNA Amplification[J].Applied Biochemistry and Biotechnology,2011,163(7):845-850.
[18] Zhang X,Lowe SB,Gooding JJ.Brief review of monitoring methods for loop-mediated isothermal amplification(LAMP)[J].Biosensors and Bioelectronics,2014,61:491-499.
计量
- 文章访问数:
- HTML全文浏览量:
- PDF下载量:
下载:
