中国动物检疫
中國動物檢疫
중국동물검역
CHINA ANMAL QUARANTINE
2015年
2期
72-77
,共6页
PRRSV%环介导等温扩增%检测方法%核酸染料%比较%结果判定
PRRSV%環介導等溫擴增%檢測方法%覈痠染料%比較%結果判定
PRRSV%배개도등온확증%검측방법%핵산염료%비교%결과판정
PRRSV%loop-mediated isothermal amplification(LAMP)%detection%nucleic acid dye%comparison%determination
本研究旨在建立一种基于颜色变化的北美洲型猪繁殖与呼吸障碍综合征病毒（PRRSV）RT-LAMP检测方法及结果判定方法，方便LAMP技术的现场检测应用。根据GenBank中美洲型PRRSV的保守序列设计一套LAMP引物，对反应进行优化后建立了PRRSV LAMP检测方法。对比Dye63、钙黄绿素（Calcein）、SYBR Green I和Picogreen 4种核酸染料对于LAMP反应的适用性。结果显示，只有Calcein、Dye63适于LAMP反应前加入，但对扩增效率均产生一定抑制，造成反应时间延迟：Calcein的延迟作用更强（约10min），Dye63延迟作用较小（约5min）。加入Dye63的LAMP反应灵敏度为2×101拷贝，而加入Calcein的灵敏度要低10倍。经多功能酶标仪对荧光强度的检测，含Dye63的反应阴阳性差异明显，可用于LAMP反应结果的判定。据此建立的基于新型核酸染料Dye63的PRRSV RT-LAMP检测方法操作简单、方便、直观，可大大减少环境污染造成的假阳性。Dye63有望成为继Calcein之后，可用于LAMP反应前加入，反应后闭管判定的一种新核酸染料，方便LAMP检测技术现场应用。
本研究旨在建立一種基于顏色變化的北美洲型豬繁殖與呼吸障礙綜閤徵病毒（PRRSV）RT-LAMP檢測方法及結果判定方法，方便LAMP技術的現場檢測應用。根據GenBank中美洲型PRRSV的保守序列設計一套LAMP引物，對反應進行優化後建立瞭PRRSV LAMP檢測方法。對比Dye63、鈣黃綠素（Calcein）、SYBR Green I和Picogreen 4種覈痠染料對于LAMP反應的適用性。結果顯示，隻有Calcein、Dye63適于LAMP反應前加入，但對擴增效率均產生一定抑製，造成反應時間延遲：Calcein的延遲作用更彊（約10min），Dye63延遲作用較小（約5min）。加入Dye63的LAMP反應靈敏度為2×101拷貝，而加入Calcein的靈敏度要低10倍。經多功能酶標儀對熒光彊度的檢測，含Dye63的反應陰暘性差異明顯，可用于LAMP反應結果的判定。據此建立的基于新型覈痠染料Dye63的PRRSV RT-LAMP檢測方法操作簡單、方便、直觀，可大大減少環境汙染造成的假暘性。Dye63有望成為繼Calcein之後，可用于LAMP反應前加入，反應後閉管判定的一種新覈痠染料，方便LAMP檢測技術現場應用。
본연구지재건립일충기우안색변화적북미주형저번식여호흡장애종합정병독（PRRSV）RT-LAMP검측방법급결과판정방법，방편LAMP기술적현장검측응용。근거GenBank중미주형PRRSV적보수서렬설계일투LAMP인물，대반응진행우화후건립료PRRSV LAMP검측방법。대비Dye63、개황록소（Calcein）、SYBR Green I화Picogreen 4충핵산염료대우LAMP반응적괄용성。결과현시，지유Calcein、Dye63괄우LAMP반응전가입，단대확증효솔균산생일정억제，조성반응시간연지：Calcein적연지작용경강（약10min），Dye63연지작용교소（약5min）。가입Dye63적LAMP반응령민도위2×101고패，이가입Calcein적령민도요저10배。경다공능매표의대형광강도적검측，함Dye63적반응음양성차이명현，가용우LAMP반응결과적판정。거차건립적기우신형핵산염료Dye63적PRRSV RT-LAMP검측방법조작간단、방편、직관，가대대감소배경오염조성적가양성。Dye63유망성위계Calcein지후，가용우LAMP반응전가입，반응후폐관판정적일충신핵산염료，방편LAMP검측기술현장응용。
The objective of the study is to establish a loop-mediated isothermal amplification assay(LAMP)for rapid detection of PRRSV and its result determination method based on nucleic acid dye in order to make the LAMP assay more convenient and fast in field application. According to the sequences published in GenBank,a set of primers were designed targeting the conserved region of PRRSV. Through optimizing the LAMP primers and reaction conditions, a LAMP assay for detection of PRRSV was established. Four nucleic acid dyes were compared for their applicability to LAMP. The result showed only Calcein and Dye63 in the four dyes could be used before the LAMP reaction but with certain inhibition to the amplification efficiency,resulting in 10 minutes reaction delay with Calcein and 5 minutes reaction delay with Dye63. In addition,the detection limit of the LAMP with Dye63 was ten times higher than with Calcein. After the reaction,the fluorescence intensity was detected by the Microplate Reader and observed with naked eye. The results showed that the negative and positive reaction presented enough differences and Dye63 was suitable for LAMP result determination. RT-LAMP was developed based on Dye63 for detection of PRRSV with simplicity, convenience,sensitivity and direct vision. And Dye63 was expected to be a new dye used in LAMP after Calcein.
