渤海湾天津沿岸海水中甲肝病毒的检测和定量

Detection and Quantification of Hepatitis A Virus in Tianjin Coastal Seawater of Bohai Bay

甲肝病毒（hepatitis A virus,HAV）是能引起传染性甲型肝炎的单链RNA病毒.用常规RT-PCR（反转录PCR）和SYBR Green实时定量RT-PCR方法,根据甲肝病毒保守的VP1-VP2基因序列设计引物,对渤海湾天津沿岸海水中的甲肝病毒进行了检测和定量分析.9个样品取自渤海湾天津塘沽以南沿岸海水,取样时间分别是2007年夏、秋、冬季和2008年春季.海水样品先用小型超滤装置（Millipore Pellicon Mini TFF）或超滤离心管（Millipore Centricon Plus-70）浓缩,然后进行RT-PCR检测.结果表明,从9个海水样品中都能扩增出192 bp的HAV cDNA,这些cDNA的核苷酸序列与GenBank中的同源序列相似性为95%-100%.用SYBR Green实时定量RT-PCR检测了春季和冬季的6个海水样品,结果表明,海水中甲肝病毒的浓度范围为5.35×10^6-4.51×10^7virus particles/L.

Hepatitis A virus （HAV） is single-strainded RNA virus that causes infectious hepatitis A. Detection and quantification of hepatitis A virus in Tianjin coastal seawater of Bohai Bay were carried out by conventional RT-PCR and SYBR Green real-time quantitative RT-PCR using the primers based on the conserved sequence at the VP1-VP2 genes of HAV. The nine samples were taken at Tianjin coastal seawater of Bohai Bay locating in the south of Tanggu, in summer, autumn and winter of 2007 and spring of 2008. For viral detection, seawater samples were concentrated either using a small ultrafiltration system （MiUipore Pellicon Mini TFF） or a Centriprep-100 centrifugal ultraf＇dtratian device （Millipore Centrieon Plus-70）. RT-PCR analysis showed that a 192 bp HAV cDNA was amplified from all nine seawater samples and the sequence identifies of these cDNAs to the homologous sequence in the GenBank were between 95% and 100%. SYBR Green real-time quantitative RT-PCR analysis indicated that HAV concentration in these samples ranged from 5.35×10^6 to 4.51 ×10^7 virus particles/L.