贝类奥尔森派琴虫可视化LAMP检测技术的建立与应用

根据环介导等温扩增(LAMP)技术原理,针对奥尔森派琴虫(Perkinsus olseni)的5.8 S核糖体RNA与内转录2间隔区序列,设计了6条特异性LAMP扩增引物,分别为两条外引物F3和B3、两条内引物FIP和BIP、两条环引物LF和LB。通过对反应条件的优化,建立了一种适用于贝类奥尔森派琴虫的可视化LAMP检测技术。该技术只对奥尔森派琴虫发生扩增反应,产生黄绿色荧光扩增产物,对其他对照DNA样品无扩增反应,不产生黄绿色荧光。对F3和B3外引物扩增产物进行测序分析,其序列与Gen-Bank中奥尔森派琴虫序列一致,检测结果具有高度特异性。对奥尔森派琴虫质粒DNA的最低检测量为100 fg。用该项LAMP技术与OIE公布的派琴虫PCR检测技术分别对50份分别来自华东及华南沿海地区的牡蛎样品进行检测,同时对PCR阳性样品进行测序分析。结果 LAMP检测到2份奥尔森派琴虫,PCR方法检测到9份派琴虫。测序分析结果表明,PCR方法检测到的9份派琴虫阳性样品中有2份为奥尔森派琴虫,其结果与LAMP检测结果相符,说明该技术适用于贝类样品奥尔森派琴虫的检测。

Establishment and Application of Visual LAMP Assay on Perkinsus olseni

Targeting the conserved region between 5.8S ribosomal RNA and internal transcribed spacer 2 on the genomic DNA of Perkinsus olseni,6 specific primers,designed based on loop mediated isothermal amplification(LAMP)assay,were listed as below: one set of outer primers F3 and B3,one set of inner primers FIP and BIP,one set of loop primers LF and LB as well.A visual LAMP method which was established to detect Perkinsus olseni following the optimized reaction conditions amplified Perkinsus olseni DNA specifically with Kelly color,whereas it did not amplify other DNA samples,thus there was no Kelly color.Sequence test was conducted to the outer primers F3 and B3,which shared the same sequence with Perkinsus olseni in GenBank.Consequently it can be deduced that this visual LAMP method was tremendously specific to Perkinsus olseni.The lowest detection dose of this visual LAMP method was 100 fg of Perkinsus olseni plasmid DNA.50 oyster samples collected from East and South coasts of China were tested by LAMP and Perkinsus PCR method offered by Office international des epizooties(OIE),simultaneously.The PCR products with amplification positive were sequenced and analyzed.The results showed that two samples were Perkinsus olseni positive by LAMP and nine samples were Perkinsus by PCR,in which two samples were Perkinsus olseni.The results from LAMP and PCR matched with each other signifying that this visual LAMP could be applied to detect Perkinsus olseni in shellfish samples.