单管半套式RT-PCR法检测贝类中轮状病毒的研究

轮状病毒(Rotavirus)是一种以贝类为载体的重要食源性病毒,目前来说,RT_PCR是贝类中轮状病毒最有效的检测方法,但通常由于病毒含量较低以及贝类中存在大量的PCR抑制物,致使将其运用于实际样本的检测时灵敏度仍较低。在实验室模拟自然环境,以人工播毒的方式使贝类富集轮状病毒,运用单管半套式RT_PCR法进行检测,并将单管半套式RT_PCR与ELISA、RT_PCR的检测灵敏度做了比较,表明单管半套式RT_PCR比ELISA的灵敏度高10 0 0倍,是传统RT_PCR的10倍,有时甚至10 0倍。另外,单管半套式RT_PCR法降低了实验过程中的内源性和外源性污染,使检测所需时间从6h缩短至4 5h ,大大改进和完善了食源性病毒检测方法。并同时以整只贝和仅以贝的消化道为样检测表明,以消化道为样时的病毒检出率和检测灵敏度较高。

Study on Rotavirus detection with single-tube seminested RT-PCR method in shellfish

Being a foodborne virus, Rotavirus was often carried by shellfish. At the present, RT_PCR was the most effective method of Rotavirus detection in shellfish, but its sensitivity was low because of low levels of virus contamination and PCR inhibitors in shellfish. So contaminated shellfish experimentally in laboratory,and imitated the natural environment to concentrate Rotavirus, then detected by the developed single_tube seminested RT_PCR. Compared with ELISA and the conventional RT_PCR, the detection sensitivity is improved by 1000 times and 10 times respectively. In addition, the outer and inner contamination is reduced dramatically and the detection time is decreased from 6h to 4.5h. When the whole shellfish and only the digestive tissues of shellfish serve as samples,the detection ratio and sensitivity are more higher when sample is the latter.