Abstract: Objective Implemented virus detection Proficiency Testing plan organized by ICLAS PEP, which contained four blind samples, and further improve laboratory testing quality. Method Analyzed and extracted nucleic acid of the samples, selected appropriate primers targeted on the promising virus to proceed polymerase chain reaction. Based on the result of polymerase chain reaction, obtain the sequencing analysis of positive result and further verify the virus in blind samples. Mixed the different dilutions of cecum contents exclude murine norovirus infection and murine norovirus culture with different concentration in the same volume, subjected to RNA extraction applied by two disparate kits and quantitative polymerase chain reaction analysis. Compared the RNA copy number derived from each experimental group with the control group. Result The four blind samples were identified as Theiler ’ s mouse encephalomyelitis virus, murine norovirus, mouse rotavirus and mouse lactate dehydrogenase hypertrophic virus separately which were agreed with the ICLAS standard result. Comparing the result of fluorescent quantitative PCR detection of norovirus in cecal contents with different dilutions, it was found that when nucleic acid was extracted with viral DNA / RNA extraction kit, the detection result showed different degrees of deviation compared with the virus control when the dilution was not higher than 1 ∶ 16, and the detection result were similar to the virus control when the dilution was 1 ∶ 32. When nucleic acid was extracted with the viral RNA extraction kit and the dilution was not less than 1 ∶ 8, the detection result was similar to the viral control. Conclusion The international comparison shows that our laboratory is reliable in virus detection, after pollution experiment research, stool samples 1 ∶ 8 diluted use viral RNA extraction kit nucleic acid for faeces, such as the best virus detection method to determine the cecum contents in samples.

Key words: ICLAS, proficiency testing, murine norovirus

摘要： 目的 通过对国际实验动物科学理事会( the International Council for Laboratory Animal Science,ICLAS)能力验证计划( Performance Evaluation Program,PEP)提供的 4 份病毒盲样进行检测,从而对实验室病毒检测能力进行验证,进一步提高实验室的病毒检测水平。 方法 对 ICLAS 提供的盲样信息进行分析,提取核酸进行 PCR 检测并对阳性产物进行测序验证。 对诺如病毒阴性盲肠内容物进行不同倍数的稀释后,与诺如病毒培养液 1 ∶ 1 混合,使用两种不同的核酸提取试剂盒进行核酸提取,通过荧光定量 PCR 方法进行诺如病毒核酸检测,比较检测的核酸拷贝数。 结果 经过检测确定 ICLAS 提供的盲样分别为小鼠脑脊髓炎病毒、小鼠诺如病毒、小鼠轮状病毒以及小鼠乳酸脱氢酶增高症病毒。 比较不同稀释度的盲肠内容物诺如病毒荧光定量 PCR 检测结果发现,使用病毒 DNA / RNA提取试剂盒提取核酸,稀释度不高于 1 ∶ 16 时,检测结果相比于病毒对照出现不同程度的偏差,稀释度为 1 ∶ 32 时检测结果与病毒对照检测结果相近。 使用病毒 RNA 提取试剂盒提取核酸,稀释度不低于 1 ∶ 8 时,检测结果与病毒对照相近。 结论 通过国际比对能力验证,说明本实验室病毒检测能力可靠,且经过污染实验研究,粪便样本1 ∶ 8 稀释后使用病毒 RNA 提取试剂盒提取核酸为粪便中诺如病毒检测的最佳预处理方法。

关键词: 国际实验动物科学协会, 能力验证, 诺如病毒