一步法微滴数字PCR检测生菜中GII型诺如病毒
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摘要
目的:采用逆转录微滴数字聚合酶链式反应(reverse transcription droplet digital polymerase chain reaction,RT-ddPCR)技术和QX200 Droplet Digital PCR System,建立一步法RT-ddPCR高灵敏快速检测生菜中GII型诺如病毒(norovirus genegroup II,NoV GII)的方法。方法:优化RT-ddPCR检测NoV GII的反应体系;通过10倍梯度稀释的NoV GII线性阳性质粒确定RT-ddPCR的检测范围;利用其他常见的肠道病毒核酸(非GII型诺如病毒)作为反应模板,与NoV GII核酸同时进行RT-ddPCR,判定反应体系的特异性,并通过不同时间多次检测样品的方式判断该检测方法的稳定性。采用ISO/TS 15216-1:2013食品中诺如病毒RNA提取方法,对人工染毒不同水平(高、中、低)的NoVGII生菜样品进行检测,同时研究去除抑制物前后对RT-ddPCR检测的影响,并与逆转录定量聚合酶链式反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)进行检测回收率的对比,探究RT-ddPCR在食品中NoV GII快速与定量检测上的发展潜力与应用前景。结果:RT-ddPCR的最高检测限为8.47×104 copies/μL,最低检测限为2.12 copies/μL,RT-ddPCR扩增效率为95.44%,标准曲线相关系数为0.997 3。在不同浓度人工染毒生菜样品中,抑制物的存在对ddPCR回收率检测结果没有显著性差异。在高、中染毒浓度下,抑制物对RT-qPCR与RT-ddPCR回收率检测结果影响不显著。低浓度下,未去除抑制物的RT-qPCR法平均回收率仅1.43%,与去除抑制物后的RT-qPCR检测结果(平均回收率为9.71%)有显著差异,且与RT-ddPCR的检测结果(未去除抑制物的RT-ddPCR平均回收率为11.80%,去除抑制物后平均回收率为12.53%)存在显著性差异。结论:生菜抑制物对RT-ddPCR的检测影响不显著,利用RT-ddPCR可有效测出低浓度的受污染样品,避免用现有的RT-qPCR方法因抑制物带来的"假阴性"检测结果。本实验建立的RT-ddPCR方法能高效、灵敏地检测出受污染食品中NoV GII的低病毒含量,在食源性病毒检测中具有可观的发展潜力和应用前景。
Objective: To develop a rapid and sensitive one-step reverse transcription droplet digital polymerase chain reaction(RT-ddPCR) assay for the detection of norovirus genogroup II(NoV GII) in lettuce. Methods: The RT-ddPCR system was optimized; the detection limit was tested using a series of 10-fold diluted enzyme digested plasmids containing the target gene sequence of NoV GII. The specificity was validated by using nucleic acids from NoV GII and other enteric viruses as targets. The repeatability of the method was also examined. Lettuce samples artificially infected with different concentrations(high, moderate, and low levels) of NoV GII were detected by RT-ddPCR following RNA extraction performed as described in ISO/TS 15216-1:2013. The influence of removal of inhibitors on the performance of RT-ddPCR was evaluated in comparison with reverse transcription quantitative polymerase chain reaction(RT-qPCR). Results: The maximum and minimum detection limits of RT-ddPCR were 8.47 × 104 and 2.12 copies/μL, respectively. The amplification efficiency was 95.44%(r = 0.997 3). The existence of inhibitors in lettuce showed no significant difference in the recovery RT-ddPCR at different contamination levels. The recovery rates of RT-qPCR at low contamination level with and without inhibitors were 1.43% and 9.71% respectively, which were significantly different from RT-ddPCR(11.80% and 12.53%respectively). Conclusions: The RT-ddPCR assay developed was stable in detecting food samples regardless of the presence of inhibitors such as lettuce. It could efficiently detect low concentrations of NoV GII virus in food without false-negative results, which may occur in RT-qPCR because of the existence of inhibitors. Therefore, this one-step RT-ddPCR assay was able to detect low levels of NoV GII in contaminated food samples, which indicates a considerable application prospect in the field of foodborne virus detection.
Objective: To develop a rapid and sensitive one-step reverse transcription droplet digital polymerase chain reaction(RT-ddPCR) assay for the detection of norovirus genogroup II(NoV GII) in lettuce. Methods: The RT-ddPCR system was optimized; the detection limit was tested using a series of 10-fold diluted enzyme digested plasmids containing the target gene sequence of NoV GII. The specificity was validated by using nucleic acids from NoV GII and other enteric viruses as targets. The repeatability of the method was also examined. Lettuce samples artificially infected with different concentrations(high, moderate, and low levels) of NoV GII were detected by RT-ddPCR following RNA extraction performed as described in ISO/TS 15216-1:2013. The influence of removal of inhibitors on the performance of RT-ddPCR was evaluated in comparison with reverse transcription quantitative polymerase chain reaction(RT-qPCR). Results: The maximum and minimum detection limits of RT-ddPCR were 8.47 × 104 and 2.12 copies/μL, respectively. The amplification efficiency was 95.44%(r = 0.997 3). The existence of inhibitors in lettuce showed no significant difference in the recovery RT-ddPCR at different contamination levels. The recovery rates of RT-qPCR at low contamination level with and without inhibitors were 1.43% and 9.71% respectively, which were significantly different from RT-ddPCR(11.80% and 12.53%respectively). Conclusions: The RT-ddPCR assay developed was stable in detecting food samples regardless of the presence of inhibitors such as lettuce. It could efficiently detect low concentrations of NoV GII virus in food without false-negative results, which may occur in RT-qPCR because of the existence of inhibitors. Therefore, this one-step RT-ddPCR assay was able to detect low levels of NoV GII in contaminated food samples, which indicates a considerable application prospect in the field of foodborne virus detection.
引文
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[26]JIA P,PURCELL M K,PAN G,et al.Analytical validation of a reverse transcriptase droplet digital PCR(RT-ddPCR)for quantitative detection of infectious hematopoietic necrosis virus[J].Journal of Virological Methods,2017,245:73-80.DOI:10.1016/j.jviromet.2017.03.010.
[27]TANG H,CAI Q,LI H,et al.Comparison of droplet digital PCRto real-time PCR for quantification of hepatitis B virus DNA[J].Bioscience,Biotechnology,and Biochemistry,2016,80(11):1-6.DOI:10.1080/09168451.2016.1196576.
[28]HUGGETT J F,FOY C A,BENES V,et al.The digital MIQEguidelines:minimum information for publication of quantitative digital PCR experiments[J].Clinical Chemistry,2013,59(6):892-902.DOI:10.1373/clinchem.2013.206375.
[29]LOISY F,ATMAR R L,GUILLON P,et al.Real-time RT-PCR for norovirus screening in shellfish[J].Journal of Virological Methods,2005,123(1):1-7.DOI:10.1016/j.jviromet.2004.08.023.
[30]KAGEYAMA T,KOJIMA S,SHINOHARA M,et al.Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR[J].Journal of Clinical Microbiology,2003,41(4):1548-1557.DOI:10.1128/JCM.41.4.1548-1557.2003.
[31]Method for quantification.International organization for Standardization.Microbiology of food and animal feed-horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR Part 1:Method of quantification:ISO/TS15216-1:2013[S].ISO,2013:9-10.
[32]BHAT S,HERRMANN J,ARMISHAW P,et al.Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number[J].Analytical and Bioanalytical Chemistry,2009,394(2):457-467.DOI:10.1007/s00216-009-2729-5.
[2]KOHO T,HUHTI L,BLAZEVIC V,et al.Production and characterization of virus-like particles and the P domain protein of GII.4 norovirus[J].Journal of Virological Methods,2012,179(1):1-7.DOI:10.1016/j.jviromet.2011.05.009.
[3]SIEBENGA J J,VENNEMA H,ZHENG D P,et al.Norovirus illness is a global problem:emergence and spread of norovirus GII.4 variants,2001-2007[J].The Journal of Infectious Diseases,2009,200(5):802-812.DOI:10.1086/605127.
[4]LU Q B,HUANG D D,ZHAO J,et al.An increasing prevalence of recombinant GII norovirus in pediatric patients with diarrhea during2010-2013 in China[J].Infection,Genetics and Evolution,2015,31:48-52.DOI:10.1016/j.meegid.2015.01.008.
[5]LU J,SUN L M,FANG L,et al.Gastroenteritis outbreaks caused by norovirus GII17,Guangdong Province,China,2014-2015[J].Emerging Infectious Diseases,2015,21(7):1240-1242.DOI:10.3201/eid2107.150226.
[6]CUI C,PAN L F,WANG Y P,et al.An outbreak of acute GII.17norovirus gastroenteritis in a long-term care facility in China:the role of nursing assistants[J].Journal of Infection and Public Health,2017,10(6):725-729.DOI:10.1016/j.jiph.2016.10.007.
[7]KAPIKIAN A Z,WYATT R G,DOLIN R,et al.Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis[J].Journal of Virology,1972,10(5):1075-1081.
[8]徐丹,陆学东.诺如病毒感染的研究进展[J].热带医学杂志,2011,11(1):109-112.
[9]TEUNIS P F M,MOE C L,LIU P,et al.Norwalk virus:how infectious is it?[J].Journal of Medical Virology,2008,80(8):1468-1476.DOI:10.1002/jmv.21237.
[10]施超,钱燕华,邵洁,等.诺如病毒性腹泻研究进展[J].江苏预防医学,2012,23(1):25-27.DOI:10.3969/j.issn.1006-9070.2012.01.010.
[11]谢雅晶,刘贤金.食源性诺如病毒在果蔬农产品中的污染及检测研究[J].病毒学报,2015,31(6):685-697.DOI:10.13242/j.cnki.bingduxuebao.002831.
[12]梁辉,蒋琦,戴光伟,等.2011-2012年广东省市售牡蛎中诺如病毒污染调查分析[J].中国食品卫生杂志,2013,25(4):359-362.DOI:10.13590/j.cjfh.2013.04.026.
[13]LE GUYADER F O S,MITTELHOLZER C,HAUGARREAU L,et al.Detection of noroviruses in raspberries associated with a gastroenteritis outbreak[J].International Journal of Food Microbiology,2004,97(2):179-186.DOI:10.1016/j.ijfoodmicro.2004.04.018.
[14]M腄E D,TRüBNER K,NEUBERT E,et al.Detection and typing of norovirus from frozen strawberries involved in a large-scale gastroenteritis outbreak in Germany[J].Food and Environmental Virology,2013,5(3):162-168.DOI:10.1007/s12560-013-9118-0.
[15]BOUWKNEGT M,VERHAELEN K,RZE疷TKA A,et al.Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains[J].International Journal of Food Microbiology,2015,198:50-58.DOI:10.1016/j.ijfoodmicro.2014.12.013.
[16]MedicalXpress.Malaysia:norovirus discovered in lettuce[EB/OL].(2012-09-13)[2017-05-13].https://medicalxpress.com/news/2012-09-malaysia-norovirus-lettuce.html.
[17]Centers for disease control and prevention.Foodborne outbreak online database(Food Tool)[DB/OL].(2016-08-25)[2017-05-13].https://wwwn.cdc.gov/foodborneoutbreaks/Default.aspx.
[18]MULLER L,RASMUSSEN L D,JENSEN T,et al.Series of norovirus outbreaks caused by consumption of green coral lettuce,denmark,April 2016[J].PLoS Currents Outbreaks,2016,8:1-7.DOI:10.1371/currents.outbreaks.115761d5d6de6a8bc7dd4b41f0f5f142.
[19]林健东,杨北兵,陈静浓.诺如病毒感染国内外研究进展[J].预防医学论坛,2010,16(8):732-734.DOI:10.16406/j.pmt.issn.1672-9153.2010.08.027.
[20]李志凯,苏国成,苏文金,等.诺如病毒检测方法研究进展[J].微生物学通报,2014,41(7):1417-1424.DOI:10.13344/j.microbiol.china.130592.
[21]XU D,WU X,HAN J,et al.Detection of GI and GII noroviruses in drinking water and vegetables using filtration and real-time RTPCR[J].European Food Research and Technology,2014,239(5):795-801.DOI:10.1007/s00217-014-2272-2.
[22]姚秀林.应用实时荧光定量RT-PCR技术快速检测基因II型诺如病毒的价值探讨[J].中国医药指南,2016,14(3):179.DOI:10.15912/j.cnki.gocm.2016.03.143.
[23]周阳,栾军,蒋鲁岩,等.实时荧光RT-PCR检测冷冻草莓中诺如病毒[J].食品安全质量检测学报,2013,4(2):515-520.
[24]BAERT L,UYTTENDAELE M,DEBEVERE J.Evaluation of viral extraction methods on a broad range of ready-to-eat foods with conventional and real-time RT-PCR for Norovirus GII detection[J].International Journal of Food Microbiology,2008,123(1/2):101-108.DOI:10.1016/j.ijfoodmicro.2007.12.020.
[25]徐文斐,房保海,林超,等.抑制剂去除对冷冻草莓中诺如病毒RTPCR检测的影响[J].食品科学,2017,38(4):296-300.DOI:10.7506/spkx1002-6630-201704048.
[26]JIA P,PURCELL M K,PAN G,et al.Analytical validation of a reverse transcriptase droplet digital PCR(RT-ddPCR)for quantitative detection of infectious hematopoietic necrosis virus[J].Journal of Virological Methods,2017,245:73-80.DOI:10.1016/j.jviromet.2017.03.010.
[27]TANG H,CAI Q,LI H,et al.Comparison of droplet digital PCRto real-time PCR for quantification of hepatitis B virus DNA[J].Bioscience,Biotechnology,and Biochemistry,2016,80(11):1-6.DOI:10.1080/09168451.2016.1196576.
[28]HUGGETT J F,FOY C A,BENES V,et al.The digital MIQEguidelines:minimum information for publication of quantitative digital PCR experiments[J].Clinical Chemistry,2013,59(6):892-902.DOI:10.1373/clinchem.2013.206375.
[29]LOISY F,ATMAR R L,GUILLON P,et al.Real-time RT-PCR for norovirus screening in shellfish[J].Journal of Virological Methods,2005,123(1):1-7.DOI:10.1016/j.jviromet.2004.08.023.
[30]KAGEYAMA T,KOJIMA S,SHINOHARA M,et al.Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR[J].Journal of Clinical Microbiology,2003,41(4):1548-1557.DOI:10.1128/JCM.41.4.1548-1557.2003.
[31]Method for quantification.International organization for Standardization.Microbiology of food and animal feed-horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR Part 1:Method of quantification:ISO/TS15216-1:2013[S].ISO,2013:9-10.
[32]BHAT S,HERRMANN J,ARMISHAW P,et al.Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number[J].Analytical and Bioanalytical Chemistry,2009,394(2):457-467.DOI:10.1007/s00216-009-2729-5.