水体中诺瓦克病毒RT-PCR检测研究

Detection of Norovirus in Water by RT-PCR

诺瓦克病毒是公认的食源性或水源性非细菌性胃肠炎的主要致病因子之一。建立诺瓦克病毒的RT-PCR检测方法,验证其特异性及灵敏度,并在实验室人工污染水样,进行模拟水样的检测,验证RT-PCR检测方法的实用性。所用引物为RNA多聚酶区的JV12/JV13,多次反复实验,均可产生327bp预期大小的特异条带,并通过杂交进一步证实了其特异性和正确性;在临床粪样中,可达到的最高检测限为50pg/mL。在共42份模拟样品的检测中,经过播毒、富集和浓缩,38份均可检出诺瓦克病毒。其中4份池塘水未检出。在模拟水样中的检测灵敏度为200pg/mL。实验中所建立的试验条件和体系可用于实际水样中诺瓦克病毒的筛选,对水质控制起到了很好的监控作用。

Norovirus is recognized as one of the major cause agents of foodbome or waterborne non-bacterial gastroenteritis. The development of RT- PCR for detection of norovirus in fecal samples, and then the potential usefulness of the assay were confirmed for detection of water samples which were contaminated by norovirus in experiment circumstance. The specificity and sensitivity were also evaluated in the assay. The anticipated band of 327bp were obtained when the primer set of JV12/JV13 were used, which targeting RNA dependent polymerase. The specific amplicous were further confirmed by southern hybridization and the same results obtained after many repeats. The detection limits were 50pg/mL in fecal samples, and 200pg/mL in artificially contaminated water samples. In a total of 42 experimentally contaminated water samples, 38 samples were positive for norovirus and 4 pond water samples were negative. The results may stem from unrecovery of norovirus and the inhibitors in these water samples. The assay developed in this study can be applied to screening norovirus in water, and can attribute to the control of water quality.