WSSV和IHHNV二重实时荧光PCR检测方法的建立
DEVELOPMENT OF A MULTIPLEX REAL2TIME PCR ASSAY FOR DETECT ION OF W SSV AND IHHNV
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摘要: 根据基因库中对虾白斑综合症病毒W SSV(AF369029)和传染性皮下及造血器官坏死病毒IHHNV(AF218226)基因序列,设计了W SSV和IHHNV的两对特异性引物和两条用不同荧光基团标记的TaqM an探针.对反应条件和试剂浓度进行优化,建立了能够同时检测W SSV和IHHNV的二重实时荧光PCR方法.该方法特异性好,对W SSV和IHHNV的检测敏感性分别达到2和20个模板拷贝数;此外抗干扰能力强,对W SSV和IHHNV不同模板浓度进行组合,仍可有效地同时检测这二个病毒.对保存的30份经常规PCR检测仅为W SSV或IHHNV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中1份为W SSV和IHHNV混合感染.本研究建立的二重实时荧光PCR方法用于W SSV和IHHNV的检测具有特异、敏感、快速、定量等优点.
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关键词:
- 白斑综合征病毒 /
- 对虾传染性皮下及造血组织坏死病毒 /
- 多重实时荧光PCR
Abstract: White spot syndrome virus (WSS V) and Infectious hypoder mal and hae matopoietic necrosis virus (I HHNV) are responsible for significant econom ic loss in the shrm i p industry1 In order to sm i ultaneously and massively identifyWSSV and I HHNV, two pairs ofprm i ers and two Taq Man probeswere designed and synthesized according to the conserved gene sequences ofWSSV (AF3690-9) and I HHNV (AF-18--6) in GenBank1The reaction parameters such as the concentra-tion oftwo pair ofprm i ers, twoTaq Man probes and the reaction bufferwere optm i ized to develop amultiplex real-tm i ePCR assay f or the rapid detection ofWSS V and I HHNV1Themultiplex real-tm i e PCR assaywas found to be specific and be able to detect and d ifferentiateWSSV and I HHNV, and no positive resu lts were observed when nucleic acid fro m Vibrio, Taura Syndro meVirus and Streptococcuswereused asmultiplex real-tm iePCR templates1The developed multiplex real-tm i ePCR assaywas compared with that ofroutinePCR1The sensitivity ofmultiplex real-tm i ePCR assaywas-and 20 template cop-ies forWSS V and I HHNV respectively, and its sensitivity was 10 3and 10-tm i es h igher than that of the routine PCR1The sa mpleswere examined using themultiplex real-tm i ePCR repeatedly and the results indicated that themultiplex real-tm i e PCR was reproducible1Different concentrations ofWSS V and I HHNV could be identified when mixed together, which miplied the assay could be applied to clin ical confir mation for smiultaneous infection ofWSSV and I HHNV1Themultiplex re-al-tmie PCR results of 30 routine PCR positive sa mples showed that one specific amplified curve was displayed when shrm i p was infected by only one of these two viral pathogens, whereas t wo specific amplified curves were displayed when shrm i p was infected by two viral pathogens1The resu lt ind icated thatmultiplex real-tmie PCR was able to detect and d iffer-entiate the presence ofeach pathogen in infected clinical shrm i p1Thismultiplex real-tm i ePCR assay is a quick, sensitive, specific and quantitative tool f or detection ofWSSV and I HHNV, and itwill be useful for the controlofWSS and I HHN in shrmip.-
Keywords:
- I HHNV /
- Mu ltiplex real-time PCR
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