Yan H Q, Wang Y Q, Cui H Y, Jin B, Gao Z Y, Wang Q Y
Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control/Beijing Research Center for Preventive Medicine, Beijing 100013, China.
Xicheng District Center for Disease Control and Prevention, Beijing 100120, China.
Zhonghua Liu Xing Bing Xue Za Zhi. 2022 Jan 10;43(1):92-97. doi: 10.3760/cma.j.cn112338-20210519-00411.
To evaluate the application of real-time RT-PCR and semi-nested RT-PCR in the detection of norovirus in oysters and analyzing the genetic characteristics of the isolates. Real-time fluorescent RT-PCR and semi-nested RT-PCR were used to detect norovirus GⅠ/GⅡ in fresh oysters collected from the markets in Beijing from November 2014 to October 2015. The detection rate of the parallel test was also analyzed. In addition, the reliability of semi-nested RT-PCR was evaluated by agreement rate and consistency test (Kappa value). The positive products of norovirus GⅠ/GⅡ capsid protein region gene by semi-nested RT-PCR were sequenced. Software BioEdit 7.0.9.0 was used for sequence alignment, and software Mega 6.0 was used to construct the evolutionary tree. In 72 samples, the detection rate of norovirus was 31.94% (23/72) by real-time RT-PCR, 38.89% (28/72) by semi-nested RT-PCR and 48.61% (35/72) by parallel test. The coincidence rate of the two methods was 73.61%, a moderate degree (Kappa value =0.43). A total of 13 norovirus strains were successfully sequenced, and 11 strains (7 GⅡ.17 strains, 2 GⅡ. 4 Sydney_ 2012 strains, 1 GⅡ. 1 strain and 1 GⅡ. 21 strain) were obtained from norovirus positive samples by two RT-PCR methods, two strains (1 GⅡ. 17 strain and 1 GⅡ. 3 strain) were obtained from real-time RT-PCR negative samples which were positive for norovirus by semi-nested RT-PCR. The similarity between these strains and reference strains from diarrhea patients, environmental sewage, and shellfish products were 84.4% - 100.0%. The parallel test of norovirus in oysters by two RT-PCR methods can improve the detection rate and detect more genotypes. Norovirus strains in oysters were highly homologous with reference strains from diarrheal patients, environmental sewage, and shellfish products. Therefore, surveillance, prevention and control for norovirus should be carried out in people who have frequent contacts with oysters and related environments.
评估实时荧光定量逆转录聚合酶链反应（RT-PCR）和半巢式RT-PCR在牡蛎中诺如病毒检测中的应用，并分析分离株的遗传特征。采用实时荧光RT-PCR和半巢式RT-PCR检测2014年11月至2015年10月从北京市场采集的新鲜牡蛎中的诺如病毒GⅠ/GⅡ型。分析平行检测的检出率。此外，通过符合率和一致性检验（Kappa值）评估半巢式RT-PCR的可靠性。对经半巢式RT-PCR扩增得到的诺如病毒GⅠ/GⅡ型衣壳蛋白区域基因的阳性产物进行测序。使用BioEdit 7.0.9.0软件进行序列比对，使用Mega 6.0软件构建进化树。在72份样本中，实时荧光RT-PCR检测诺如病毒的检出率为31.94%（23/72），半巢式RT-PCR为38.89%（28/72），平行检测为48.61%（35/72）。两种方法的符合率为73.61%，为中等程度（Kappa值=0.43）。共成功测序13株诺如病毒，通过两种RT-PCR方法从诺如病毒阳性样本中获得11株（7株GⅡ.17型、2株GⅡ.4悉尼_2012型、1株GⅡ.1型和1株GⅡ.21型），从实时荧光RT-PCR阴性但半巢式RT-PCR诺如病毒阳性的样本中获得2株（1株GⅡ.17型和1株GⅡ.3型）。这些毒株与腹泻患者、环境污水和贝类产品中的参考毒株的相似度为84.4% - 100.0%。两种RT-PCR方法对牡蛎中诺如病毒进行平行检测可提高检出率并检测到更多基因型。牡蛎中的诺如病毒毒株与腹泻患者、环境污水和贝类产品中的参考毒株高度同源。因此，应对经常接触牡蛎及相关环境的人群开展诺如病毒的监测、防控工作。
