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目的建立一种多重PCR结合液相芯片技术同时检测A型轮状病毒、GI型诺如病毒和GII型诺如病毒的方法。方法建立多重PCR扩增方法,将PCR产物与偶联核酸探针的微球混合物进行杂交,利用Luminex 200液相芯片检测仪来检测杂交结果。对建立的A型轮状病毒、GI型诺如病毒和GII型诺如病毒液相芯片检测方法进行灵敏度、干扰性分析,并应用该方法对68份粪便样品的核酸进行检测。结果灵敏性实验结果表明,该方法对GII型诺如病毒的检测灵敏度较高,检出限为10 pg/PCR;对轮状病毒的检出限为100 pg/PCR;该方法对GI型诺如病毒的检测灵敏度较低,检出限为1 ng/PCR。干扰性实验结果表明,轮状病毒、GI型诺如病毒和GII型诺如病毒的液相芯片检测,当病毒之间的模板量不同时(1∶1、1∶2、2∶1、1∶5、5∶1、1∶10、10∶1、1∶100、100∶1),仍能够准确检测;对68份临床粪便样品的核酸进行检测,检测出A型轮状病毒样品19份,GI型诺如病毒样品1份,GII型诺如病毒样品18份;轮状病毒和GII型诺如病毒混合感染1份,GI和GII型诺如病毒混合感染1份。结论本研究建立的轮状病毒和诺如病毒液相芯片检测方法通过一次检测能同时检测A型轮状病毒、GI型诺如病毒和GII型诺如病毒,有助于诊断病毒引起的急性腹泻。
Objective To establish a multiplex PCR combined with liquid-phase microarray technique for the simultaneous detection of rotavirus A, GI Norovirus and Norovirus GII. Methods A multiplex PCR method was established to hybridize the PCR product with the microsphere mixture with the nucleic acid probe. The hybridization results were detected by Luminex 200 liquid-phase chip detector. The sensitivity and interference analysis of established detection methods for rotavirus A, norovirus GI and Norovirus GII were performed, and the nucleic acid of 68 stool samples was detected by this method. Results The results of sensitivity test showed that the method was sensitive to GII norovirus and the detection limit was 10 pg / PCR. The detection limit of rotavirus was 100 pg / PCR. If the detection sensitivity of the virus is low, the detection limit is 1 ng / PCR. The results of the interference experiment showed that liquid phase microarray detection of rotavirus, Norovirus GI and Norovirus GII showed that when the amount of the template was different between viruses (1: 1, 1: 2, 2: 1, 1 : 5,5: 1, 1:10, 10: 1, 1: 100, 100: 1), and still be able to detect accurately; 68 nucleic acid samples of clinical stool samples were tested and 19 samples of type A rotavirus , 1 sample of Norovirus type GI, 18 samples of Norovirus type GII, 1 mixed rotavirus and Norovirus GII, and 1 mixed GI and GII Norovirus. Conclusion The detection method of rotavirus and norovirus liquid-phase microarray established in this study can detect A virus, GI norovirus and GII Norovirus at the same time, which can be used to diagnose virus-induced acute diarrhea .