【摘要】 目的感染冠状病毒的动物向环境排毒主要是通过粪便,建立直接从粪便样品对动物冠状病毒进行检测的分子技术具有重要的公共卫生学意义。方法通过计算机模拟和实验方法对已报道的2对针对冠状病毒pol基因的通用引物的通用性进行了验证。不经传统的病毒分离,直接从环境样品中提取病毒RNA,通过一步法RT-PCR进行检测,并通过分子杂交和NestedPCR扩增结合TaqMan探针实时荧光检测的PCR技术,提高对冠状病毒检测的灵敏度和准确度,并对猪、禽冠状病毒感染的临床样品进行分析检测。结果2对引物可以覆盖所有已知的冠状病毒,包括SARS,RT-PCR产物通过测序可以确定冠状病毒种类;实时荧光定量NestedPCR有很高的灵敏度,可以灵敏地检出所有供试的阳性样品,而荧光增量实时监测可以排除凝胶电泳检查的假阳性。结论该研究为从环境中普查和鉴定冠状病毒提供了可靠的技术方法。更多还原

【Abstract】 Objective The coronavirus infected animals discharge infectious viral particles into the environment via feces. A molecular approach for detecting all the coronavirus in one test from fecal samples had significance for public health. Methods The universality of two primer pairs, which were previously described as universal primers for detection of pol gene of coronavirus, were validated both by using simulated-PCR and wet experi -ments. To improve the sensitivity and reliability of the detection methods, a protocol integrating RT-PCR amplication, molecular hybridization and real-time TaqMan nested-PCR were developed and validated based on analysis of standard samples and unknown clinical samples from pigs and chickens. Results The two pairs of primers covered all known coronavirus (including SARS), and the amplified DNA fragments also had the capacity to discriminate coronavirus species. Real-time TaqMan nested-PCR approach provided highly sensitive detection of the positive samples, and excluded false positive detection. [WTHZ]Conclusion The approach developed in this study was sensitive and reliable in detection and identification of the coronavirus from complicated environmental samples such as animal feces.更多还原