Encinas-García Trinidad, Mendoza-Cano Fernando, Enríquez-Espinoza Tania, Luken-Vega Leonardo, Vichido-Chávez Rodrigo, Sánchez-Paz Arturo
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Laboratorio de Referencia, Análisis y Diagnóstico en Sanidad Acuícola, Hermosa 101, Col. Los Ángeles, Hermosillo, Sonora C.P. 83106, Mexico.
Departamento de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Av. Colosio s/n, entre Sahuaripa y Reforma, Hermosillo, Sonora 83000, Mexico.
J Virol Methods. 2015 Feb;212:53-8. doi: 10.1016/j.jviromet.2014.10.014. Epub 2014 Nov 8.
The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.
斑节对虾浓核病毒（PstDV）（也称为传染性皮下和造血组织坏死病毒，IHHNV）是主要的对虾病原体之一，在全球范围内的养殖和野生对虾种群中均有分布。IHHNV的爆发已造成重大经济损失，并带来负面社会影响。由于PstDV基因组的几个部分可能已内源性整合到斑节对虾的核基因组中，目前的诊断性PCR检测可能会产生假阳性结果。为此开发了一种基于一步法qPCR SYBR Green的定量实时聚合酶链反应（qPCR）检测方法，用于检测对虾样本中的不同IHHNV分离株。该检测方法的检测限为每个反应81个靶向DNA病毒拷贝。通过熔解曲线分析评估了该检测方法的特异性，结果表明IHHNV产物在81.4±0.044°C处产生单一熔解峰。该检测方法比传统PCR更灵敏。标准化的PCR显示出高度灵敏、特异、稳健且可重复，这使其成为用于IHHNV诊断应用和一般研究的经济且强大的工具。
