IHHNV和LCDV实时定量PCR检测方法的建立与应用-水产品工与贮藏工程专业毕业论文.docx

测，共检测到12批LCDV阳性样品，并利用标准曲线对病毒进行了定量分析。 测，共检测到12批LCDV阳性样品，并利用标准曲线对病毒进行了定量分析。 将阳性样品的常规PCR产物进行纯化测序，证实是LCDV序列。Taqman实时定 量PCR检测LCDV，灵敏度高，特异性好，可以进行定量分析，可成为一种早 期检测LCDV的新方法。 SYBR Green I实时定量PCR中，优化了定量PCR的反应条件，反应体系的 最佳引物浓度为0．51xM。该方法能特异检测出淋巴囊肿病毒，不能扩增流行性造 血器官坏死病毒(Epizootic haematopoietic necrosis virus，EHNV)、虎纹蛙病毒 (Tiger frog virus，TFV)、蛙虹彩病毒(Bohle iridovirus，BIV)、新加坡石斑虹彩 病毒(Singapore grouper iridovirus，SGIV)、鳜鱼传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus，[SKNV)及真鲷虹彩病毒(Sea bream iridovirus， RSⅣ)。利用该方法对12份发病牙鲆鱼样品进行检测，均呈阳性。重复性测试 中，SYBR Green I实时定量PCR检测LCDV质粒DNA显示出很好的批内重复 性和批间重复性。建立的SYBR Green I实时定量PCR方法用于LCDV的检测 具有成本低、特异、灵敏、重复性好等优点，同样可成为一种早期检测LCDV 的新方法。 关键词：传染性皮下及造血器官坏死病毒(m如呵V)；淋巴囊肿病毒(LCDv)； 实时定量PCR；检测；测序 V App App I i cat i on of rea I-t i me quant i tat i ve PeR assay for l HHNV and LCDV Abstract Infeedous hypodermal and hematopoietic necrosis virus(mmrv)Call infect worldwide cultured shrimp and caLLsC serious economic loss to the shrimp culture industry．The experiment surveyed 84 Litopenaeus vannamei samples from Guangxi Province with real-time quantitative PCR(mqPCR)for the fi st time，with the conventional PCR as a contrast．The positive rate of rt-qPCR was 79．8％and that of the conventional PCR was 40．5％，and each sample positive by conventional PeR was also positive by rt—qPCR indicating that the infection rate of Litopenaeus vannamei with IHHNV in Guangxi Province was hi曲．Nucleotide sequencing Was performed on 30 samples that were IHHNV positive．The results indicated that t11ey were IHHNV sequence when analysed with the soft package of DNA STAR and compared with the GenBank using the NCBI Blast program．The sequences of the 30 samples were conservative谢tll variation on two positions and classified to 4 types．rt-qPCR Can be all effective diagnose tool for detecting IHHNV infections on shrimp． LCDV Was widely distributed in worldwide freshwater,brackish water and seawater and could infect more than 140 species of fishes belonging to 9 orders，34 families．LCD was