CAJ | 학술논문

以cfb基因保守序列的6个区域设计4条特异性引物,利用链置换DNA聚合酶( BstDNA polymer：ase)在恒温(65℃)下保温40 min,建立了无乳链球菌 Streptococcus agalactiae 的 LAMP 技术。建立的LAMP方法能够特异性地检测无乳链球菌,检测灵敏度为3·7×101~3·7×102 cfu/mL,反应前加入显色剂钙黄绿素避免二次污染。现场应用中采用FTA卡采集红尾皇冠鱼Aequidens rivulatus病鱼肝脏组织病原菌,操作简便、快捷。研究表明,针对无乳链球菌cfb基因建立的LAMP检测方法具有较高的特异性及稳定性,尤其是具有快速、简便、成本低等优点,可用于无乳链球菌的野外现场快速检测。
이cfb기인보수서렬적6개구역설계4조특이성인물,이용련치환DNA취합매( BstDNA polymer：ase)재항온(65℃)하보온40 min,건립료무유련구균 Streptococcus agalactiae 적 LAMP 기술。건립적LAMP방법능구특이성지검측무유련구균,검측령민도위3·7×101~3·7×102 cfu/mL,반응전가입현색제개황록소피면이차오염。현장응용중채용FTA잡채집홍미황관어Aequidens rivulatus병어간장조직병원균,조작간편、쾌첩。연구표명,침대무유련구균cfb기인건립적LAMP검측방법구유교고적특이성급은정성,우기시구유쾌속、간편、성본저등우점,가용우무유련구균적야외현장쾌속검측。
The cfb gene of Streptococcus agalactiae was amplified by a set of four specific primers that recognize six distinct sequences of the target in 40 min by incubating all of the reagents in a single tube with BstDNA polymerase at a constant 65 ℃. A loop-mediated isothermal amplification ( LAMP) assay was developed and validated for the specific detection of Streptococcus agalactiae, with detection limits of 3. 7í101-3. 7í102 cfu/mL. The resulting am-plicons were visualized by adding calcein to the reaction tube before the reaction. FTA cards was used for collection and purification of nucleic acids from a liver of green terror Aequidens rivulatus with pathogenic bacteria in field ap-plications. The findings demonstrate that the LAMP-based assay is a sensitive and reliable means for the detection of Streptococcus agalactiae with rapidity, simplicity and low cost, especially suitable for on-site rapid diagnosis.