| 严寒秋,王永全,崔海洋,靳博,高志勇,王全意.应用2种RT-PCR方法检测和分析北京市市场销售牡蛎中诺如病毒基因特征[J].中华流行病学杂志,2022,43(1):92-97 |
| 应用2种RT-PCR方法检测和分析北京市市场销售牡蛎中诺如病毒基因特征 |
| Application of two RT-PCR methods for detection of norovirus in market-sold oysters and norovirus genetic characteristic analysis, a survey conducted in Beijing |
| 收稿日期:2021-05-19 出版日期:2022-01-26 |
| DOI：10.3760/cma.j.cn112338-20210519-00411 |
| 中文关键词: 诺如病毒 实时荧光反转录聚合酶链式反应 半巢式反转录聚合酶链式反应 基因特征 牡蛎 |
| 英文关键词: Norovirus Real-time RT-PCR Semi-nested RT-PCR Genetic characteristics Oyster |
| 基金项目:首都卫生发展科研专项（2020-2-1011） |
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| 中文摘要: |
| 目的 应用实时荧光RT-PCR和半巢式RT-PCR方法检测和分析牡蛎中诺如病毒的基因特征。方法 应用实时荧光RT-PCR和半巢式RT-PCR方法，对2014年11月至2015年10月北京市采集的新鲜市售牡蛎进行诺如病毒GⅠ/GⅡ组并联试验检测，分析检出率，应用符合率和一致性检验（Kappa值）对半巢式RT-PCR方法进行可靠性评价，应用半巢式RT-PCR方法扩增诺如病毒GⅠ/GⅡ衣壳蛋白区基因，对阳性产物进行测序。采用BioEdit 7.0.9.0软件进行序列比对、Mega 6.0软件构建进化树。结果 72份样品中，实时荧光RT-PCR、半巢式RT-PCR和并联试验的诺如病毒检出率分别为31.94%（23/72）、38.89%（28/72）和48.61%（35/72）。2种方法符合率为73.61%，中度一致（Kappa值=0.43）。测序成功13株诺如病毒，11株（7株GⅡ.17、2株GⅡ.4 Sydney_2012、1株GⅡ.1和1株GⅡ.21基因型）为2种RT-PCR方法检测诺如病毒阳性样本所得；2株（GⅡ.17和GⅡ.3基因型各1株）为半巢式RT-PCR方法检测诺如病毒阳性样本、实时荧光RT-PCR方法检测诺如病毒阴性样本所得。这些毒株与腹泻患者、环境污水和贝类水产品参考株的相似性为84.4%~100.0%。结论 用2种RT-PCR并联法检测牡蛎中诺如病毒，不仅能提高检出率还能获得更多基因型；牡蛎中诺如病毒毒株与人源、环境污水及贝类水产品参考毒株高度同源，应对经常接触牡蛎的人群及相关环境进行诺如病毒监测和防控。 |
| 英文摘要: |
| Objective 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.Methods 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.Results 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%.Conclusions 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. |
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