Article(id=1241035816504455888, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241035810628235909, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202407091, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1720454400000, receivedDateStr=2024-07-09, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773815595984, onlineDateStr=2026-03-18, pubDate=1733760000000, pubDateStr=2024-12-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773815595984, onlineIssueDateStr=2026-03-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773815595984, creator=13701087609, updateTime=1773815595984, updator=13701087609, issue=Issue{id=1241035810628235909, tenantId=1146029695717560320, journalId=1227665162245664772, year='2024', volume='51', issue='23', pageStart='4225', pageEnd='4416', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773815594584, creator=13701087609, updateTime=1773815743629, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1241036435843764756, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241035810628235909, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1241036435843764757, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1241035810628235909, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4392, endPage=4397, ext={EN=ArticleExt(id=1241035817070686950, articleId=1241035816504455888, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Analysis of urban sewage SARS-CoV-2 monitoring results in Guiyang city in 2023, columnId=1228016571713520579, journalTitle=Modern Preventive Medicine, columnName=Health Supervison, runingTitle=null, highlight=null, articleAbstract=
Objective

To investigate the concentration and genetic variations of SARS-CoV-2 in sewage in Guiyang city from February to December 2023.

Methods

A total of 1 034 sewage samples were analyzed for ORF1ab and N gene concentrations using quantitative PCR, and full viral genome sequencing was performed using the Illumina sequencing platform.

Results

Among the 1 034 samples collected, 587 were tested positive for SARS-CoV-2. The median concentration of the ORF1ab gene was 3.69 copies/ml, and the median concentration of the N gene was 7.57 copies/ml. There was a correlation between the concentration of SARS-CoV-2 in sewage and the number of reported cases from medical institutions. Full genome sequencing was conducted on five SARS-CoV-2 positive samples with CT values <32, achieving an average coverage ranging from 63.91% to 97.28%. All viral types identified were Omicron sub lineages, with a total of 110 mutation sites identified. The S protein exhibited 22 amino acid changes, including the critical mutation E484A.

Conclusion

The detection rate of SARS-CoV-2 in urban sewage in Guiyang city is relatively high, and the viral concentration correlates well with clinical cases. The application of whole genome sequencing technology allows for the detection of genetic variants in the viral material present in sewage and identifies multiple mutation sites. Monitoring viruses in urban sewage effectively captures the spatiotemporal trends of the disease, serving as an important complement to clinical testing and providing a reference for local governments to implement precise epidemic prevention policies.

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目的

了解贵阳市2023年2—12月污水中新型冠状病毒(SARS-CoV-2)含量和基因变异情况。

方法

采用荧光定量PCR方法完成了1 034份污水样本的ORF1ab基因和N基因浓度的检出,使用Illumina测序平台完成病毒全基因组测序。

结果

在收集到的1 034份样本中,检测出587份SARS-CoV-2阳性样本,ORF1ab基因浓度中位数为3.69拷贝/ml,N基因浓度中位数为7.57拷贝/ml,污水中SARS-CoV-2的浓度与医疗机构报告病例数之间存在相关性。对5份Ct<32的SARS-CoV-2阳性样本利用测序平台完成了全基因组测序,平均覆盖度在63.91%~97.28%之间,病毒分型均为奥密克戎亚分支,共鉴定出110个突变位点,S蛋白共鉴定出22个氨基酸的改变,其中包括关键突变E484A。

结论

贵阳市城市污水中新型冠状病毒检出率较高,病毒浓度与临床病例具有良好相关性,应用全基因组测序技术,检测污水中病毒遗传物质的变异株组成情况,并鉴别出多个变异位点,通过对城市污水病毒监测,能有效地对疾病的时空趋势进行把握,是临床检测的重要补充,为地方政府实施精确疫情防控政策提供参考。

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袁飞,E-mail:
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黄金金(1999—),女,硕士在读,研究方向:实验室病原监测

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黄金金(1999—),女,硕士在读,研究方向:实验室病原监测

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Water Research, 2023, 244: 120444., articleTitle=Wastewater genomic sequencing for SARS-CoV-2 variants surveillance in wastewater-based epidemiology applications, refAbstract=null)], funds=[Fund(id=1241069126538556231, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, awardId=gzwkj-2024-491, language=CN, fundingSource=2024年度贵州省卫生健康委科学技术基金项目(gzwkj-2024-491), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1241069117483053404, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, xref=1., ext=[AuthorCompanyExt(id=1241069117487247710, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, companyId=1241069117483053404, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=School of Public Health and Health, Guizhou Medical University, Key Laboratory of Environmental Pollution and Disease Monitoring, Ministry of Education, Guiyang, Guizhou 561113, China), AuthorCompanyExt(id=1241069117495636319, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, companyId=1241069117483053404, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1.贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵州 贵阳 561113)]), AuthorCompany(id=1241069117604688226, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, xref=2., ext=[AuthorCompanyExt(id=1241069117608882532, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, companyId=1241069117604688226, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.贵阳市疾病预防控制中心,贵州 贵阳 550018)])], figs=[ArticleFig(id=1241069124781142771, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=EN, label=Figure 1, caption=Temporal distribution of SARS-CoV-2 ORF1ab target gene and SARS-CoV-2 N target gene concentrations in sewage samples, figureFileSmall=AurhI1rFeymWxu8r5PA2JQ==, figureFileBig=ESeVGzfDbZJSryGkeQOTIQ==, tableContent=null), ArticleFig(id=1241069124906971897, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=CN, label=图1, caption=污水样本中新冠病毒ORF1ab靶基因和N靶基因浓度的时间分布, figureFileSmall=AurhI1rFeymWxu8r5PA2JQ==, figureFileBig=ESeVGzfDbZJSryGkeQOTIQ==, tableContent=null), ArticleFig(id=1241069125032801026, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=EN, label=Figure 2, caption=Comparison of ORF1ab gene concentration, N gene degree concentration, and number of cases in sewage samples from Guiyang city, China, figureFileSmall=7x9h6GryhBLVi60E50G2Xg==, figureFileBig=2y1F3ilh9RZqUaf/4+YvPQ==, tableContent=null), ArticleFig(id=1241069125255099146, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=CN, label=图2, caption=贵阳市污水样本中ORF1ab基因浓度、N基因度浓度与病例数比较, figureFileSmall=7x9h6GryhBLVi60E50G2Xg==, figureFileBig=2y1F3ilh9RZqUaf/4+YvPQ==, tableContent=null), ArticleFig(id=1241069125330596624, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=EN, label=Figure 3, caption=Comparison of typing composition of SARS-CoV-2 variants in five sewage sequencing samples in Guiyang city in 2023, figureFileSmall=rxJvM6tfMD7bY05mDLKAEg==, figureFileBig=9s0h8L63vPgRbPsyDWXXAA==, tableContent=null), ArticleFig(id=1241069125393511192, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=CN, label=图3, caption=贵阳市2023年5份污水测序样本SARS-CoV-2变异株分型构成比, figureFileSmall=rxJvM6tfMD7bY05mDLKAEg==, figureFileBig=9s0h8L63vPgRbPsyDWXXAA==, tableContent=null), ArticleFig(id=1241069125502563101, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=EN, label=Table 1, caption=

Detection of SARS-CoV-2 in sewage samples in Guiyang city from February to December of 2023

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月份检测份数检出[n(%)]靶基因浓度中位数(拷贝/ml)
ORF1abN
27739(50.65)2.5711.32
39957(57.58)3.3612.23
48846(52.27)3.944.47
59979(79.80)6.5717.87
69971(71.72)6.2812.07
79950(50.51)3.127.23
89955(55.56)4.629.57
98849(55.68)5.717.88
109955(55.56)2.184.80
119942(42.42)2.403.68
128844(50.00)2.816.20
合计1 034587(56.77)3.697.57
), ArticleFig(id=1241069125620003618, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=CN, label=表1, caption=

贵阳市2023年2—12月污水样本新冠病毒检出情况

, figureFileSmall=null, figureFileBig=null, tableContent=
月份检测份数检出[n(%)]靶基因浓度中位数(拷贝/ml)
ORF1abN
27739(50.65)2.5711.32
39957(57.58)3.3612.23
48846(52.27)3.944.47
59979(79.80)6.5717.87
69971(71.72)6.2812.07
79950(50.51)3.127.23
89955(55.56)4.629.57
98849(55.68)5.717.88
109955(55.56)2.184.80
119942(42.42)2.403.68
128844(50.00)2.816.20
合计1 034587(56.77)3.697.57
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Correlation analysis of ORF1ab gene concentration and N Gene concentration with sampling day wastewater treatment volume and population served by wastewater treatment plants

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靶基因服务人口数量采样日污水处理量
ORF1ab浓度相关系数rs0.033-0.032
P0.4480.463
N浓度相关系数rs0.0300.035
P0.4820.418
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ORF1ab基因浓度、N浓度与采样日污水处理量和污水处理厂服务人口相关性分析

, figureFileSmall=null, figureFileBig=null, tableContent=
靶基因服务人口数量采样日污水处理量
ORF1ab浓度相关系数rs0.033-0.032
P0.4480.463
N浓度相关系数rs0.0300.035
P0.4820.418
), ArticleFig(id=1241069126018462512, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=EN, label=Table 3, caption=

Whole genome sequencing results of 5 wastewater samples in Guiyang city

, figureFileSmall=null, figureFileBig=null, tableContent=
污水处理厂采样日期ORF1ab基因浓度(拷贝/ml)N基因浓度(拷贝/ml)平均覆盖深度平均覆盖度(%)Pangolin分型
D厂2023/2/912.9448.24220.4563.91BA.2.86.1、BA.2.86.3、BF.7.14、XBB.1.16、XBB.1.24.3、XBB.1.5
I厂2023/2/911.457.22688.4964.62BA.2.86.3、BF.7.14、BN.1.2、FR.1、XBB.1.24.3
E厂2023/2/917.960.211 291.4981.04BA.2.86.3、BF.7.14、BN.1.2、FR.1
B厂2023/6/1227.6286.487 568.796.88BF.7.14、BN.1.2、FL.2.4、HK.2、JG.3、XBB.1.16.1、XBB.1.24.3、XBB.1.5
J厂2023/6/1246.23172.639 049.8897.28BF.7.14、BN.1.2、FL.2.4、HK.2、JG.3、JJ.1、XBB.1.16.1、XBB.1.24.3、XBB.1.5
), ArticleFig(id=1241069126119125812, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=CN, label=表3, caption=

贵阳市5份污水样本全基因组测序结果

, figureFileSmall=null, figureFileBig=null, tableContent=
污水处理厂采样日期ORF1ab基因浓度(拷贝/ml)N基因浓度(拷贝/ml)平均覆盖深度平均覆盖度(%)Pangolin分型
D厂2023/2/912.9448.24220.4563.91BA.2.86.1、BA.2.86.3、BF.7.14、XBB.1.16、XBB.1.24.3、XBB.1.5
I厂2023/2/911.457.22688.4964.62BA.2.86.3、BF.7.14、BN.1.2、FR.1、XBB.1.24.3
E厂2023/2/917.960.211 291.4981.04BA.2.86.3、BF.7.14、BN.1.2、FR.1
B厂2023/6/1227.6286.487 568.796.88BF.7.14、BN.1.2、FL.2.4、HK.2、JG.3、XBB.1.16.1、XBB.1.24.3、XBB.1.5
J厂2023/6/1246.23172.639 049.8897.28BF.7.14、BN.1.2、FL.2.4、HK.2、JG.3、JJ.1、XBB.1.16.1、XBB.1.24.3、XBB.1.5
), ArticleFig(id=1241069126215594809, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=EN, label=Table 4, caption=

Amino acid changes of SARS-CoV-2 virus in five sequenced samples from Guiyang city, China

, figureFileSmall=null, figureFileBig=null, tableContent=
样本编号S基因氨基酸变化
D厂2.9L452R;E484A;ΔH69/V70
I厂2.9L452R;E484A;F486V;C1243E;ΔH69/V70
E厂2.9L452R;E484A;F486V;C1243E;ΔH69/V70
B厂6.12Q52H;V83A;H146Q;Q183E;G257S;G339R;L68I;V445L;V445A;G446S;N460K;F486L;F486A;F490S
J厂6.12V83A;H146Q;Q183E;G339R;L368I;V445L;V445A;G446S;N460K;F486L;F486S;F490S
), ArticleFig(id=1241069126320452417, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1241035816504455888, language=CN, label=表4, caption=

贵阳市5份测序样本中SARS-CoV-2病毒氨基酸变化情况

, figureFileSmall=null, figureFileBig=null, tableContent=
样本编号S基因氨基酸变化
D厂2.9L452R;E484A;ΔH69/V70
I厂2.9L452R;E484A;F486V;C1243E;ΔH69/V70
E厂2.9L452R;E484A;F486V;C1243E;ΔH69/V70
B厂6.12Q52H;V83A;H146Q;Q183E;G257S;G339R;L68I;V445L;V445A;G446S;N460K;F486L;F486A;F490S
J厂6.12V83A;H146Q;Q183E;G339R;L368I;V445L;V445A;G446S;N460K;F486L;F486S;F490S
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贵阳市2023年1 034份城市污水新型冠状病毒监测结果分析
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黄金金 1 , 倪德航 1 , 徐素芹 1 , 周建松 2 , 简洁 2 , 洪峰 1 , 袁飞 1, 2 , 蒋家俊 2
现代预防医学 | 卫生监督 2024,51(23): 4392-4397
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现代预防医学 | 卫生监督 2024, 51(23): 4392-4397
贵阳市2023年1 034份城市污水新型冠状病毒监测结果分析
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黄金金1, 倪德航1, 徐素芹1, 周建松2, 简洁2, 洪峰1, 袁飞1, 2 , 蒋家俊2
作者信息
  • 1.贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵州 贵阳 561113
  • 2.贵阳市疾病预防控制中心,贵州 贵阳 550018
  • 黄金金(1999—),女,硕士在读,研究方向:实验室病原监测

通讯作者:

袁飞,E-mail:
Analysis of urban sewage SARS-CoV-2 monitoring results in Guiyang city in 2023
Jin-jin HUANG1, De-hang NI1, Su-qin XU1, Jian-song ZHOU2, Jie JIAN2, Feng HONG1, Fei YUAN1, 2 , Jia-jun JIANG2
Affiliations
  • School of Public Health and Health, Guizhou Medical University, Key Laboratory of Environmental Pollution and Disease Monitoring, Ministry of Education, Guiyang, Guizhou 561113, China
出版时间: 2024-12-10 doi: 10.20043/j.cnki.MPM.202407091
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目的

了解贵阳市2023年2—12月污水中新型冠状病毒(SARS-CoV-2)含量和基因变异情况。

方法

采用荧光定量PCR方法完成了1 034份污水样本的ORF1ab基因和N基因浓度的检出,使用Illumina测序平台完成病毒全基因组测序。

结果

在收集到的1 034份样本中,检测出587份SARS-CoV-2阳性样本,ORF1ab基因浓度中位数为3.69拷贝/ml,N基因浓度中位数为7.57拷贝/ml,污水中SARS-CoV-2的浓度与医疗机构报告病例数之间存在相关性。对5份Ct<32的SARS-CoV-2阳性样本利用测序平台完成了全基因组测序,平均覆盖度在63.91%~97.28%之间,病毒分型均为奥密克戎亚分支,共鉴定出110个突变位点,S蛋白共鉴定出22个氨基酸的改变,其中包括关键突变E484A。

结论

贵阳市城市污水中新型冠状病毒检出率较高,病毒浓度与临床病例具有良好相关性,应用全基因组测序技术,检测污水中病毒遗传物质的变异株组成情况,并鉴别出多个变异位点,通过对城市污水病毒监测,能有效地对疾病的时空趋势进行把握,是临床检测的重要补充,为地方政府实施精确疫情防控政策提供参考。

污水流行病  /  新型冠状病毒  /  城市污水  /  基因测序  /  变异
Objective

To investigate the concentration and genetic variations of SARS-CoV-2 in sewage in Guiyang city from February to December 2023.

Methods

A total of 1 034 sewage samples were analyzed for ORF1ab and N gene concentrations using quantitative PCR, and full viral genome sequencing was performed using the Illumina sequencing platform.

Results

Among the 1 034 samples collected, 587 were tested positive for SARS-CoV-2. The median concentration of the ORF1ab gene was 3.69 copies/ml, and the median concentration of the N gene was 7.57 copies/ml. There was a correlation between the concentration of SARS-CoV-2 in sewage and the number of reported cases from medical institutions. Full genome sequencing was conducted on five SARS-CoV-2 positive samples with CT values <32, achieving an average coverage ranging from 63.91% to 97.28%. All viral types identified were Omicron sub lineages, with a total of 110 mutation sites identified. The S protein exhibited 22 amino acid changes, including the critical mutation E484A.

Conclusion

The detection rate of SARS-CoV-2 in urban sewage in Guiyang city is relatively high, and the viral concentration correlates well with clinical cases. The application of whole genome sequencing technology allows for the detection of genetic variants in the viral material present in sewage and identifies multiple mutation sites. Monitoring viruses in urban sewage effectively captures the spatiotemporal trends of the disease, serving as an important complement to clinical testing and providing a reference for local governments to implement precise epidemic prevention policies.

Sewage epidemiology  /  SARS-CoV-2  /  Urban sewage  /  Gene sequencing  /  Variants
黄金金, 倪德航, 徐素芹, 周建松, 简洁, 洪峰, 袁飞, 蒋家俊. 贵阳市2023年1 034份城市污水新型冠状病毒监测结果分析. 现代预防医学, 2024 , 51 (23) : 4392 -4397 . DOI: 10.20043/j.cnki.MPM.202407091
Jin-jin HUANG, De-hang NI, Su-qin XU, Jian-song ZHOU, Jie JIAN, Feng HONG, Fei YUAN, Jia-jun JIANG. Analysis of urban sewage SARS-CoV-2 monitoring results in Guiyang city in 2023[J]. Modern Preventive Medicine, 2024 , 51 (23) : 4392 -4397 . DOI: 10.20043/j.cnki.MPM.202407091
新型冠状病毒(severe acute respiratory syndrome coronavirus,SARS-CoV-2)是引起新型冠状病毒感染的病原体。据世界卫生组织统计,截至2024年2月,全球累积超过7.7亿确诊病例[1]。新冠病毒在世界造成大流行,并存在多种变异体,这些突变主要是由于病毒刺突S糖蛋白的多次改变所引起[2]。在五个“关切变异株”中,奥密克戎的传播力和免疫逃逸能力显著增强,在2022年初代替其他变异体成为优势流行变异株[3-4]。新型冠状病毒主要通过呼吸道飞沫和密切接触传播,部分患者会出现腹泻症状,病毒颗粒通过粪便脱落,在呼吸道样本中不能检测出新冠病毒后,仍能通过粪便检测[5-6]。2020年,澳大利亚首次报道在污水中检测到新冠病毒[7],随后,研究人员将基于污水的流行病学(wastewater-based epidemiology, WBE)应用于新型冠状病毒的监测,且可量化污水中的新冠病毒浓度推断污水采样覆盖范围人群的疫情情况,从而调整防控措施[8]。部分区域将基因组测序纳入污水监测中,美国的一项研究将污水中新型冠状病毒的单核苷酸变异(single nucleotide polymorphism, SNV)与临床患者基因组测序中检测到的单核苷酸变异进行了比较,确定了超过一半的单核苷酸变异是在全球范围内未在临床样本中发现的新型单核苷酸变异[9];奥地利的一项全国范围的研究通过污水样本推断时空丰度[10],并可实现在临床样本之前识别新的变异体[11]。为了探究城市污水中新冠病毒监测对覆盖区域人群疾病流行情况的反映效果,本研究分析了2023年2—12月贵阳市主城区11座污水处理厂的1 034份污水样本的新型冠状病毒核酸监测结果。
为确保采样方法的一致性,本研究选取贵阳市主城区11座拥有自动采样器的污水处理厂进行污水样本的收集,自2023年2月9日起,每周两次从贵阳市的11座污水处理厂收集24 h(早上10点至次日早上10点)混合样本,每小时采样100 ml,共2 400 ml。
从贵阳市各个医疗机构获取2023年2—12月内贵阳市新型冠状病毒感染病例情况。
样本参照WS/T799-2022《污水中新型冠状病毒富集浓缩和核酸检测方法标准》中铝盐混凝沉淀法进行前处理:将50 ml的污水样本于4℃、2 500×g条件下进行预离心30 min后,留取上清液;预离心后的上清液转移至100 ml螺口锥形瓶中,加入0.5 ml 0.3 mol/L的氯化铝溶液,用1 mol/L氢氧化钠溶液或1 mol/L盐酸溶液调节水样pH=(6.0±0.1)。将螺口锥形瓶盖拧紧,置于恒温振荡培养箱中,以150 r/min的速度在室温下混合15 min后,在4 ℃、1 900×g离心5 min;弃除离心管中的上清液,在剩余胶体中加入(0.20 g±0.01) g乙二胺四乙酸二钠二水合物,摇晃数十次至胶体变为液态,转移至10 ml离心管中。将10 ml离心管置于水浴锅中,60 ℃水浴10 min后得到富集浓缩的污水样本。
富集浓缩后的污水样本利用天隆全自动核酸提取仪提取核酸,然后根据武汉明德生物科技新型冠状病毒2019-nCoV核酸检测试剂盒的操作步骤对核酸样本进行荧光PCR检测,根据荧光PCR扩增曲线和试剂盒说明书结果判读标准对结果进行判定。用新型冠状病毒核酸基因组标准物质制备标准曲线,通过荧光定量RT-PCR检测获得Ct值,以Ct值为横坐标、浓度的对数值为纵坐标绘制标准曲线,获得扩增效率和R2。扩增效率在90%~110%范围内,且R2≥0.99时,标准曲线有效。用荧光定量RT-PCR 检测污水核酸样本,将获得的Ct值带入标准曲线,计算核酸样本中新冠病毒的浓度,并根据实验操作过程将核酸样本中新冠病毒浓度换算成污水样本中每份样本的ORF1ab基因浓度和N基因浓度。
将Ct值<32的核酸样本进行进一步测序。根据ULSEN®超灵敏度新冠病毒全基因组捕获试剂盒的操作步骤对新冠病毒全基因组进行逆转录和扩增,扩增完成的核酸加入磁珠进行纯化,利用Qubit Tube对样本的浓度进行测定,根据NexteraXT DNAw文库制备试剂盒说明完成DNA文库构建。上机试剂盒为MiSeq v3,在Illumina测序平台完成病毒全基因组测序。
采用SPSS 23进行数据分析,率的比较采用χ2分析,率的两两比较采用Bonferroni法,利用Spearman法进行相关性分析,检验水准α= 0.05。全基因测序结果通过杭州柏熠科技的分子溯源遗传分析系统上完成新冠病毒全基因组的序列的拼接、质量评估,并序列比对,分析核酸变异、氨基酸变异和完成基因分型(pangolin分型)。
2023年2—12月共收集11座污水处理厂污水样本1 034份,对样本富集浓缩后进行实时荧光RT-PCR检测,11座污水处理厂SARS-CoV-2总检出率为56.77%,SARS-CoV-2 ORF1ab基因浓度中位数为3.69拷贝/ml,N基因浓度中位数为7.57拷贝/ml。11座污水处理厂(A、B、C、D、E、F、G、H、I、J、K)检出率分别为43.62%、68.09%、52.13%、48.94%、72.34%、58.51%、55.32%、54.26%、71.28%、61.70%、38.30%,各个污水处理厂之间检出率差异具有统计学意义(χ2=46.493,P<0.001)。不同污水处理厂间检出率进行两两比较,调整检验水准α'=0.000 9,6座污水处理厂(C、D、F、G、H、J)SARS-CoV-2检出率差异没有统计学意义;B污水处理厂、E污水处理厂、I再生水厂与、A污水处理厂、K污水处理厂SARS-CoV-2检出率差异具有统计学意义。见表1
整个研究期间,各个月均有SARS-CoV-2检出,其中5月份检出率最高,为79.8%,其次是6月,检出率为71.72%;11月检出率最低,为42.42%。不同月份之间检出率差异具有统计学意义(χ2=44.021,P<0.001),各月份间检出率进行两两比较,调整检验水准α'=0.000 9,5月、6月检出率差异无统计学意义,5月与其余9个月差异有统计学意义;6月与11月检出率差异有统计学意义。病毒ORF1ab基因浓度与N基因浓度变化趋势大致相同,2—12月污水中新冠病毒浓度变化存在多个高峰,在第35周(8月第4周)达到高峰。见图1
通过对新冠病毒的ORF1ab浓度和N浓度分别与采样日污水处理量和污水处理厂服务人口进行Spearman秩相关分析,发现采样日污水处理水量和污水处理厂服务人口数量对新冠病毒在污水中的检出浓度不存在相关性。见表2
在研究期间,根据Spearman相关性分析结果显示,污水处理厂样本ORF1ab基因浓度与N基因浓度表现出显著相关(rs=0.781,P<0.001),ORF1ab基因浓度与医疗机构报告病例数之间存在正相关关系(rs=0.441,P<0.05),N基因浓度与医疗机构报告病例数之间存在正相关关系(rs=0.482,P<0.001)。N基因浓度与医疗机构报告病例数之间的相关性略强于ORF1ab基因浓度的。污水中病毒基因浓度与临床病例数变化趋近一致。见图2
本研究共收集587份阳性样本,对于Ct值<32的5份阳性样本进行了全基因组测序。5份样本平均覆盖深度为220.45~9 049.88,平均覆盖度为63.91%~97.28%;pangolin分型结果均为奥密克戎变异株,2月份三份样本占比最高的变异株为BF.7.14,6月份两份样本占比最高的变异株为FL.2.4,在同一月份不同污水厂之间的变异株构成不同。见表3图3
在完成污水测序的5份样本中共鉴定出110个突变位点,非同义突变占比67.27%,涉及了11个编码区的突变(E、N、M、ORF10、ORFab、ORF3a、ORF6、ORF7ab、基因间隔区ORF7ab-ORF8、ORF8、S),发生突变最多的基因是S基因和ORF1ab基因,S基因突变占38.18%,ORF1ab基因突变占比33.64%。SARS-Cov-2的感染能力主要取决于刺突表面蛋白(S蛋白),刺突表面蛋白发生突变可能影响单克隆抗体的中和反应,从而变异体发生免疫逃逸在人群中传播[12]。本研究中S基因共鉴定出42个变异位点,引起22个氨基酸的改变,在改变的氨基酸中,其中4个来自对蛋白质相互作用至关重要的17个关键氨基酸。见表4
在疫情常态化的背景下,本研究通过对贵阳市2023年2—12月1 034份城市污水样本中新型冠状病毒的监测,结合临床数据,了解人群中疫情的流行情况和变异情况。与之前奥地利、加拿大的研究相似[13-14],污水中病毒的浓度与医院报告病例数关联性强,无论是ORF1ab基因浓度还是N基因浓度都与医疗机构报告的病例呈现良好的相关性。自从2023年1月起中国新冠病毒调整为 “乙类乙管”,强制性的核酸检测取消,临床样本变得稀缺,本研究的临床样本来自于贵阳市6个主城区的医疗机构报病卡信息,很大程度上反应的是症状相对严重需要到医院就医人群的情况,污水样本的监测比临床样本更容易发现无症状感染者和轻症感染者,对城市污水样本的监测成了掌握疫情情况的重要补充。不同城市污水处理厂之间的阳性检出率不同,有报道称为SARS-Cov-2浓度与废水相关参数(PH、电导率、BOD5、COD、NH4、总氮、总磷、TSS)[15]呈现弱相关,本研究显示,新型冠状病毒浓度与采样日的进水流量和服务人口之间也不存在关联,侧面反映污水处理厂的规模大小对新冠病毒的浓度影响度小,针对不同规模的污水样本SARS-CoV-2的检测可以有效的反映不同区域的流行的变化,这对将污水流行病应用于社区的精确疫情控制提供了科学的理论依据。
奥密克戎毒株是目前SRAS-Cov-2传播力最强的变异株,2021年11月首次在南非发现[16]。本研究中5份测序样本发现的变异株均为奥密克戎毒株,2月份占比最高的是BF.7.4毒株,六月份转为FL.2毒株,主要流行变异毒株的改变,引起了一波新的流行,这体现在5月、6月医疗机构报告病例的上升,与之对应时间内,污水样本阳性检出率升高,病毒拷贝数升高。在同一采样时间,不同污水处理厂的测序样本变异株的构成比并不相同,表明对污水样本中新冠病毒的混合变异体的测序可以反映区域中变异株的流行情况,实现精准疫情防控。在2月份的3份测序样本中,从BF.7.4毒株中鉴定出5个S基因的突变,有1个(F486V)是蛋白质相互作用关键氨基酸。研究表明,E484是RBD上的重要残基,已有研究证明E484的氨基酸替换为K、Q、P可以降低中和效价,发生E484A的突变,提示免疫逃逸能力增强[17],2月9日的3份污水测序样本中均检测到了E484A突变。有研究证明L452R突变可以降低几种单克隆抗体和中和反应,增加了感染风险[18],在2月的3份污水样本中均检测到L452R突变,此外与病毒复制增加有关的H69/V70的缺失突变[19]也在污水样本中检测到。根据污水中SARS-CoV-2的突变信息,监测关键突变位点,有助于预测污水处理厂覆盖人群疾病感染风险。根据国家监测数据显示,2023年6月主要流行毒株为XBB系列EG.5.1.1,但在贵阳城市污水中,6月的2份测序样本显示,占比最高的属于FL.2.4毒株,与当时流行的主要毒株并不相同,这也说明了在临床样本缺少测序数据的情况下,污水样本全基因组测序结果实现对地方疾病流行情况做出精确的掌握的能力。
本研究中能达到测序要求的污水样本较少,587份阳性水样中仅完成了5份样本的测序,且有2份样本的平均覆盖度小于80%,这与污水样本的特性有关,污水中病毒浓度较低,污水样本中的复杂基质可能干扰或者抑制PCR,在污水中的病毒一般是碎片化的状态,测序通量对基因的覆盖度也有影响[20],这些原因限制了污水样本的有效测序。
本研究监测了贵阳市2023年2—12月,1 034份城市污水样本,描述了污水中新型冠状病毒浓度与医疗机构报告数的相关性,此外,发现新型冠状病毒浓度与采样日的进水流量和服务人口之间不存在关联,由此说明城市污水中新冠病毒的监测对覆盖区域人群新冠病毒流行情况反映效果良好。通过将污水中新型冠状病毒载量的监测和全基因测序监测结合起来能够有效的、及时的、全面的发现污水采样点覆盖范围人群新冠病毒的流行情况、流行的主要变异株以及变异位点情况,根据突变信息预测流行趋势,及时的为当地政府实施精准的疫情控制政策提供参考。
  • 2024年度贵州省卫生健康委科学技术基金项目(gzwkj-2024-491)
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2024年第51卷第23期
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doi: 10.20043/j.cnki.MPM.202407091
  • 接收时间:2024-07-09
  • 首发时间:2026-03-18
  • 出版时间:2024-12-10
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  • 收稿日期:2024-07-09
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2024年度贵州省卫生健康委科学技术基金项目(gzwkj-2024-491)
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    1.贵州医科大学公共卫生与健康学院,环境污染与疾病监控教育部重点实验室,贵州 贵阳 561113
    2.贵阳市疾病预防控制中心,贵州 贵阳 550018

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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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