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【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】 To establish a rapid, specific and sensitive TaqMan-LNA probe for detection of IHHNV in shrimp by real-time fluorescence quantitative PCR, which provides a simple and convenient detection technique for IHHNV clinical diagnosis and epidemic situation monitoring. 【Method】 Based on the published sequences of IHHNV in GenBank, specific primers and TaqMan-LNA probes were designed based on the conserved region of IHHNV. The conditions of fluorescence quantitative PCR were optimized to shorten the detection time and improve the specificity and sensitivity. TaqMan LNA probe fluorescence quantitative PCR was used to detect 300 samples of Litopenaeus vannamei in Guangxi and to verify the practicability of the method. 【Result】 The results showed that the sensitivity of TaqMan-LNA probe to detect IHHNV was high, about 10 virions / reaction with a wide range of quantitation, and had a good linear relationship in the range of 1.6 × 101-1.6 × 108 copies / μL. Which showed no specific response to DNA of SPV and DNA of V. parahaemolyticus and RNA of TSV, with the coefficient of variation of 0.53% ~ 1.75% and the coefficient of variation of 0.81% -1.14% Repeatability and reproducibility were both good and the results were stable and reliable.The results of clinical application showed that 193 samples were positive and the positive rate was 64.3% in 300 samples of Litopenaeus vannamei, indicating IHHNV infection of Litopenaeus vannamei cultured in coastal area of Guangxi Higher rate. 【Conclusion】 Fluorescence quantitative PCR for detection of IHHNV by TaqMan-LNA probe is rapid, specific, sensitive, reproducible and quantitative. It can be used to detect IHHNV in shrimp.