Belák S
Joint Research and Development Division of the National Veterinary Institute, OIE Collaborating Centre for the Application of Polymerase Chain Reaction Methods for Diagnosis of Viral Diseases in Veterinary Medicine,Uppsala, Sweden.
Dev Biol (Basel). 2007;128:103-12.
The highly contagious transboundary animal diseases (TADs), e.g., foot-and-mouth disease (FMD), classical swine fever (CSF), African swine fever (ASF) and highly pathogenic avian influenza (HPAI) are regularly occurring and re-occurring on various continents, causing severe losses. This epidemiological situation indicates the urgent need for the development of powerful, robust and high capacity new diagnostic methods in order to detect and identify the causative agents very rapidly. This report is on the experiences of an OIE Collaborating Centre and those of the MULTIPLEX-PCR and the LAB-ON-SITE EU project consortia with the deveopment of novel methods for the improved molecular diagnosis of a range of viral diseases. Thermal amplification based real-time PCR methods (e.g.,TaqMan, Molecular Beacons, Primer-Probe Energy Transfer, and Light Upon eXtension (LUX) fluorogenic primers), and amplification without thermocycling have been elaborated for the improved diagnosis of TADs, such as FMD, swine vesicular disease, vesicular stomatitis, CSF, ASF, HPAI and Newcastle disease (ND). The simultaneous detection of various pathogens in a disease complex is facilitated by the development of multiplex PCR packages. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the molecular diagnostic procedures have become rapid, robust and automated. Quality control is strengthened by special precautions to avoid false positive and false negative results. By following the steps of OIE standardisation and validation, the diagnostic PCR assays have become nationally and nternationally standardised and harmonised. The development of additional methods, like padlock probes and microarrays, is further improving the arsenal of nucleic acid based novel molec ular diagnostic tests for TADs.
高传染性跨界动物疾病（TADs），如口蹄疫（FMD）、经典猪瘟（CSF）、非洲猪瘟（ASF）和高致病性禽流感（HPAI），在各大洲经常发生且反复出现，造成严重损失。这种流行病学情况表明，迫切需要开发强大、可靠且高容量的新诊断方法，以便快速检测和鉴定病原体。本报告介绍了一个世界动物卫生组织（OIE）协作中心以及多重聚合酶链反应（MULTIPLEX-PCR）和现场实验室欧盟项目联盟在开发一系列病毒疾病改进分子诊断新方法方面的经验。基于热扩增的实时聚合酶链反应方法（如TaqMan、分子信标、引物-探针能量转移和延伸发光（LUX）荧光引物）以及无热循环扩增方法已被精心设计用于改进TADs的诊断，如口蹄疫、猪水疱病、水疱性口炎、经典猪瘟、非洲猪瘟、高致病性禽流感和新城疫（ND）。多重聚合酶链反应组合的开发有助于在疾病复合体中同时检测各种病原体。通过引入核酸提取和移液机器人技术，以及多通道实时聚合酶链反应仪，分子诊断程序变得快速、可靠且自动化。通过采取特殊预防措施避免假阳性和假阴性结果，加强了质量控制。遵循OIE标准化和验证步骤后，诊断性聚合酶链反应检测在国内和国际上实现了标准化和协调一致。锁式探针和微阵列等其他方法的开发进一步完善了用于TADs的基于核酸的新型分子诊断测试手段。
