摘要
【目的】建立快速、特异、灵敏检测对虾传染性皮下及造血组织坏死病毒(IHHNV)的TaqMan-LNA探针荧光定量PCR方法,为IHHNV的临床诊断和疫情监测提供更简便的检测技术。【方法】根据GenBank已发表的IHHNV毒株序列,在IHHNV保守区序列设计特异性引物和TaqMan-LNA探针;对荧光定量PCR反应条件进行优化以缩短检测时间,提高特异性和灵敏度;应用TaqMan-LNA探针荧光定量PCR对广西地区的300份凡纳滨对虾样品进行检测,验证方法的实用性。【结果】TaqMan-LNA探针荧光定量PCR检测IHHNV的灵敏度高,约为10个病毒粒子/反应,定量范围宽,在1.6×101～1.6×108拷贝/μL的范围内具良好的线性关系;与SPF凡纳滨对虾组织、WSSV、副溶血弧菌的DNA和TSV的RNA均无反应,特异性好;组内变异系数为0.53%～1.75%,组间变异系数为0.81%～1.14%,重复性和重现性均较好,结果稳定可靠;临床应用结果表明,300份凡纳滨对虾样品检出193份阳性,阳性率为64.3%,表明广西沿海地区养殖的凡纳滨对虾IHHNV感染率较高。【结论】利用TaqMan-LNA探针建立检测IHHNV的荧光定量PCR方法具有快速、特异、灵敏、重复性好、能定量等优点,可用于对虾IHHNV的检测。
[Objective]The present experiment was conducted to develop a TaqMan-LNA probe real-time PCR assay for detection of infectious hypodermal and hematopoietic necroses virus（IHHNV） to provide convenient detection technology for clinical diagnosis and epidemic monitoring of IHHNV.[Method]The specific primers and TaqMan-LNA probes were designed from conserved IHHNV sequences obtained from GenBank.The real-time PCR assay was optimized to reduce the detection time and to improve the sensitivity and specificity.Three hundred clinical shrimp samples collected from Guangxi were detected by real-time PCR to evaluate practicality of assay.[Result]The real-time PCR assay accurately detected and quantified IHHNV.It showed better linear relationship from 1.6×101 to 1.6×108 copies/μL and had a minimum detection limit of about 10 virion.The method was specific for IHHNV and had no cross-reaction with three other shrimp pathogen such as white spot syndrome virus（WSSV） ,Vibrio parahaemolyticus,taura syndrome virus（TSV） .This method also showed excellent reproducibility with the coefficient of variations ranging from 0.53-1.75% within group and 0.81-1.14% among different groups.Out of 300 samples,193（64.3%） showed positive results for IHHNV which revealed that IHHNV infection in Litopenaeus vannanei is a major threat for shrimp cultivation in Guangxi.[Conclusion]The TaqMan-LNA probe real-time PCR assay is a quick,sensitive,specific,quantitative and reproducible tool for detecting IHHNV.
出处
《南方农业学报》
CAS
CSCD
2011年第12期1545-1549,共5页
Journal of Southern Agriculture
基金
广西直属公益性科研院所基本科研业务费专项项目(2060302GXIF-2009-05)
