Wang Chi-Young J, Giambrone Joseph J, Smith Bruce F
Department of Poultry Science, College of Veterinary Medicine, Auburn University, Auburn, Alabama 36849, USA.
J Clin Microbiol. 2002 Jul;40(7):2584-90. doi: 10.1128/JCM.40.7.2584-2590.2002.
Duck hepatitis B virus (DHBV) belongs to the Hepadnaviridae family, which includes human Hepatitis B virus (HBV) and Woodchuck hepatitis virus. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-microl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was >or=4 degrees C. The optimal condition for storage was -70 degrees C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.
鸭乙型肝炎病毒（DHBV）属于嗜肝DNA病毒科，该科还包括人类乙型肝炎病毒（HBV）和土拨鼠肝炎病毒。由于先天性传播，它在野生和家养鸭中广泛分布。HBV是一个全球性的健康问题，携带者有发展为肝硬化和肝癌的风险。由于其传染性，与HBV打交道的医护人员和科学家必须接种疫苗。由于DHBV与HBV相似，它是HBV的安全替代物。在滤纸上采集血清和血液样本已被用于筛查代谢紊乱、遗传疾病和病毒感染以及基因组的进化研究。在本研究中，通过PCR检测滤纸上干燥的血清和血液中的DHBV。0.1微升的样本就足以进行检测。研究了滤纸对DHBV的固定潜力，观察到使用Whatman滤纸时PCR产物的产量最高。干燥血清在不同储存温度下4周内保持稳定，但当温度≥4℃时，PCR产物的产量会下降。最佳储存条件是-70℃。本文应用了一种新开发的基于监测荧光增加来定量PCR的方法，该荧光增加是由SYBR绿染料与双链产物结合引起的，用于监测扩增情况。克隆到质粒中的DHBV基因组DNA用于生成定量PCR的标准DHBV DNA。它在DHBV颗粒拷贝数方面验证了PCR结果。通过熔解曲线分析证明了PCR的特异性，并基于由GC含量和序列确定的熔解温度，证明了从干燥血清中扩增的两种DHBV分离株的差异。它比其他基于PCR的DNA技术更简便。使用滤纸上干燥的血清可以将来自远距离地区且存在冷藏和运输问题的样本邮寄到诊断实验室。样本可以存档以供比较，并用作克隆和测序的DNA来源。
