Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups Ⅰ and Ⅱ

Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three Taq Man real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit(assay A:Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays(assay B: Light Cycler RNA Master Hybprobe and assay C: Real Time ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity(103 DNA copies/m L) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups.No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle(Cq) value of assay B for GII was lower than assays A and C with statistical significance(P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17,and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

Genomic surveillance of coxsackievirus A10 reveals genetic features and recent appearance of genogroup D in Shanghai,China,2016–2020

Coxsackievirus A10(CVA10)is one of the major causative agents of hand,foot and mouth disease(HFMD).To investigate the epidemiological characteristics as well as genetic features of CVA10 currently circulating in Shanghai,China,we collected a total of 9,952 sporadic HFMD cases from January 2016 to December 2020.In the past five years,CVA10 was the fourth prevalent causatives associated with HFMD in Shanghai and the overall positive rate was 2.78%.The annual distribution experienced significant fluctuations over the past five years.In addition to entire VP1 sequencing,complete genome sequencing and recombination analysis of CVA10 isolates in Shanghai were further performed.A total of 64 near complete genomes and 11 entire VP1 sequences in this study combined with reference sequences publicly available were integrated into phylogenetic analysis.The CVA10sequences in this study mainly belonged to genogroup C and presented 91%-100%nucleotide identity with other Chinese isolates based on VP1 region.For the first time,our study reported the appearance of CVA10 genogroup D in Chinese mainland,which had led to large-scale outbreaks in Europe previously.The recombination analysis showed the recombination break point located between 5,100 nt and 6,700 nt,which suggesting intertypic recombination with CVA16 genogroup D.To conclusion,CVA10 genogroup C was the predominant genogroup in Shanghai during 2016-2020.CVA10 recombinant genogroup D was firstly reported in circulating in Chinese mainland.Continuous surveillance is needed to better understand the evolution relationships and transmission pathways of CVA10 to help to guide disease control and prevention.

多重荧光RT-PCR同时检测GⅠ型和GⅡ型诺如病毒方法的建立

目的建立可同时检测GⅠ型和GⅡ型诺如病毒的双重荧光RT-PCR快速检测方法,并应用于临床标本的检测。方法使用针对GI型和GⅡ型诺如病毒各亚型代表株保守序列设计的特异性引物和TaqMan探针,通过调整引物、探针浓度优化最佳反应条件,建立一步法荧光RT-PCR快速检测反应体系。对该方法的灵敏度、特异性、稳定性进行评估,并对临床粪便标本进行检测,与已有的单重荧光RT-PCR方法进行比较。结果实验结果表明,该检测方法特异性强,对GⅠ型、GⅡ型诺如病毒、轮状病毒、肠道腺病毒、甲型肝炎病毒进行检测,均无交叉反应;对GⅠ型和GⅡ型诺如病毒核酸的最低检出限分别可达1pg/mL和10pg/mL,并且重复性好;对101份临床标本分别用已有的单重荧光RT-PCR和本文建立的多重荧光RT-PCR进行检测,结果显示本文建立的多重荧光RT-PCR方法对GⅠ型诺如病毒的检测能力优于前者。结论本研究建立的多重荧光RT-PCR方法能同时对GⅠ型和GⅡ型诺如病毒进行快速检测,并且灵敏度高,特异性好,可用于感染性腹泻暴发中诺如病毒的快速检测。

济南地区婴幼儿腹泻病杯状病毒的流行特征

目的阐明山东济南地区婴幼儿腹泻中杯状病毒感染的流行状况及其特征。方法收集济南市儿童医院婴幼儿病毒性腹泻粪便标本,使用杯状病毒特异性引物对标本进行RT-PCR检测。将杯状病毒阳性PCR产物纯化后,进行克隆和序列分析,使用BioEdit和MEGA软件与GenBank中的参考株进行核苷酸序列同源性分析并绘制系统进化树。结果在212例病毒性腹泻标本中,杯状病毒RT-PCR阳性62例,检出率为29.25%。经序列分析,所有杯状病毒均为诺如病毒基因组Ⅱ型。结论杯状病毒是山东地区婴幼儿腹泻病的主要病原之一,目前以诺如病毒基因组Ⅱ型毒株流行为主。

苏州市14起诺如病毒胃肠炎暴发疫情流行特征分析

目的了解苏州市诺如病毒胃肠炎暴发疫情的流行病学特征。方法对2010-2014年发生的14起诺如病毒胃肠炎暴发疫情进行描述性流行病学分析,采用聚合酶链反应对病例肛拭子、粪便、呕吐物、水、环境涂抹标本进行核酸检测。结果 2011-2014年,苏州市共报告14起诺如病毒胃肠炎暴发疫情,罹患率波动范围为0.89%~21.18%。14起疫情中12起（85.71%）发生于托幼机构和中小学校,医院、农村各1起。全年均有发生,但以寒冷季节发生较多。仅1起查明与二次供水受到污染有关,13起未证实暴发原因。核酸检测结果12起由诺如病毒GII基因组引起,2起由GI基因组引起。结论诺如病毒已成为苏州市急性胃肠炎暴发疫情的重要病原,托幼机构和学校是高发场所,GII基因组是主要基因组。在暴发疫情处理中,应加强传染源和传播途径的调查。

猪细环病毒两种基因型双重PCR检测方法的建立

为建立检测猪细环病毒两种基因型(PTTV1和PTTV2)的双重PCR方法,本研究根据GenBank中PTTV1、PTTV2的UTR基因序列,设计合成了2对特异引物,并通过对扩增条件的筛选,建立了PTTV1和PTTV2的双重PCR检测方法。该方法可以同时扩增PTTV1的324 bp和PTTV2的522 bp特异性片段,而扩增猪圆环病毒2型和猪细小病毒DNA结果均为阴性,对PTTV1和PTTV2的最低检出量分别为100 copies和10 copies。该方法适合对PTTV1和PTTV2的联合检测。

中国部分地区猪细环病毒1型和2型的分子检测

细环病毒(TTV)是近年来发现的一种新型人畜共患DNA病毒,广泛存在于包括人类和家畜在内的多种哺乳动物中。目前我国猪群中TTV的流行病学报道较少。为研究猪细环病毒(PTTV)在我国的流行情况,采用聚合酶链反应(PCR)对2009年我国9个省市部分发病猪场中的PTTV1和PTTV2进行检测。结果显示,191份病料中PTTV的总阳性率为77.0%(147/191),其中PTTV1的单一阳性率为68.6%(131/191),PTTV2的单一阳性率为53.9%(103/191),两基因型的共感染率为45.5%(87/191)。进一步分析发现,在不同猪场、不同年龄猪群、不同病料组织中,PTTV的感染情况均不相同。此外,对41份健康猪的血清进行检测,结果显示,PTTV总阳性率、PTTV1单一阳性率、PTTV2单一阳性率及两基因型的共感染率分别为43.9%(18/41)、36.6%(15/41)、24.4%(10/41)和12.2%(5/41)。结果提示,发病猪体内PTTV总阳性率、PTTV1单一阳性率、PTTV2单一阳性率及两基因型的共感染率均显著高于健康猪(P<0.01)。PTTV在临床上是否对猪致病或与其他病原有协同作用,需进一步研究。

Acute gastroenteritis outbreak caused by a GII.6 norovirus

AIM:To report an acute gastroenteritis outbreak caused by a genogroup 2 genotype 6(GII.6) strain norovirus in Shanghai,China.METHODS:Noroviruses are responsible for approximately half of all reported gastroenteritis outbreaks in many countries.Genogroup 2 genotype 4 strains are the most prevalent.Rare outbreaks caused by GII.6 strains have been reported.An acute gastroenteritis outbreak occurred in an elementary school in Shanghai in December of 2013.Field and molecular epidemiologic investigations were conducted.RESULTS:The outbreak was limited to one class in an elementary school located in southwest Shanghai.The age of the students ranged from 9 to 10 years.The first case emerged on December 10,2013,and the last case emerged on December 14,2013.The cases peaked on December 11,2013,with 21 new cases.Of 45 students in the class,32 were affected.The main symptom was gastroenteritis,and 15.6%(5/32) of the cases exhibited a fever.A field epidemiologic investigation showed the pathogen may have been transmitted to the elementary school from employees in a delicatessen via the first case student,who had eaten food from the delicatessen one day before the gastroenteritis episodes began.A molecular epidemiologic investigation identified the cause of the gastroenteritis as norovirus strain GII.6;the viral sequence of the student cases showed 100% homology with that of the shop employees.Genetic relatedness analyses showed that the new viral strain is closely related to previously reported GII.6 sequences,especially to a strain reported in Japan.CONCLUSION:This is the first report to show that norovirus strain GII.6 can cause a gastroenteritis outbreak.Thus,the prevalence of GII.6 noroviruses requires attention.

2006年上海监测点婴幼儿腹泻标本中诺如病毒基因分析

[目的]了解2006年上海监测点婴幼儿病毒性腹泻散发病例中诺如病毒基因型别及核酸变异情况。[方法]应用一步法RT-PCR扩增89份病毒性腹泻标本中诺如病毒核酸,测序后应用基因分析软件包对核酸序列进行分析。[结果]12条核酸序列经测序和比对均属于诺如病毒GII基因组,其中3条核酸测序序列与2006年德国公布的序列同源性相近;其余9条核酸测序序列与2004年中国、2004、2005年日本、2006年荷兰公布的序列同源性相近。[结论]2006年上海地区婴幼儿中存在诺如病毒GII基因组散发病例,并以GII4基因型为主。

荧光定量RT-PCR检测诺如病毒基因Ⅱ型方法的建立和评估

目的建立灵敏、特异的诺如病毒基因Ⅱ型的荧光定量RT-PCR方法,用于临床腹泻粪便样本病毒的定量检测。方法构建质粒DNA,并转录合成RNA作为标准品,建立和优化诺如病毒基因Ⅱ型荧光定量RT-PCR方法和反应体系,评价该方法的灵敏度、特异性、重复性,并进行临床粪便样本的检测。结果此方法最低可以检出102拷贝数/μl,能特异地检出诺如病毒基因Ⅱ型,与诺如病毒基因Ⅰ型无交叉反应,与柯萨奇病毒B组、脊髓灰质炎病毒、肠道病毒、星状病毒、甲肝病毒、埃可病毒和轮状病毒无交叉反应。针对标准品,2次批内试验的荧光信号循阈值(Ct值)变异系数(CV)分别为1.60%、0.70%,2次批间试验的变异系数(CV)分别为0.40%、0.40%,均在5%以下。结论本研究建立的实时定量RT-PCR检测诺如病毒基因Ⅱ型的方法,其灵敏度、特异性和重复性良好,可用于该病毒的快速检测。

诺沃克样病毒广州地区分子流行病学分析

目的根据广州市多起由诺沃克样病毒引起的食物中毒患者粪便标本中检测到的Ⅱ型诺沃克样病毒序列,初步了解广州地区诺沃克样病毒的分子流行病学情况。方法用RT-PCR对病人粪便中的诺沃克样病毒核酸进行扩增,将阳性产物测序,并与GenBank中的序列相比较。结果归纳出4个代表性序列,并且相似率都不高。结论我国存在诺沃克样病毒引起的食物中毒,而其RNA复制酶的保守区段变异普遍较大。

上海地区部分婴幼儿腹泻标本中诺如病毒基因分析

目的了解2006年上海哨点医院婴幼儿疑似病毒性腹泻散发病例中诺如病毒基因型别和基因特征。方法应用反转录-聚合酶链反应(RT-PCR)扩增标本中诺如病毒核酸,测序后使用DNAstar基因分析软件与Genbank中的参考株进行核酸序列分析。结果13份测序结果均属于诺如病毒GⅡ遗传组,以GⅡ.4基因型为主。其中2份核酸序列与2006年德国汉堡毒株序列同源性相近,分别为86.2%和88.2%;2份核酸序列与2006年荷兰乌得勒支毒株序列同源性相近,均为83.9%;其余9份核酸序列与2004年中国广西、2004年和2005年日本东京毒株序列同源性相近,为87.3%～97.5%。结论2006年上海哨点医院婴幼儿疑似病毒性腹泻标本中存在诺如病毒GⅡ.4基因型散发病例,不同病毒株之间差异较大。

Clinical and epidemiological characteristics of norovirus gastroenteritis among hospitalized children in Lebanon

AIM To assess the burden of norovirus(No V) and to determine the diversity of circulating strains among hospitalized children in Lebanon. METHODS Stool samples were collected from children presenting with acute gastroenteritis to six major hospitals in Lebanon. A total of 739 eligible stool samples, testing negative for diarrhea caused by rotavirus as a possible viral pathogen, were collected between January 2011 and June 2013. A standardized questionnaire including demographic, epidemiological and clinical observations was used at the time of hospitalization of children presenting with diarrhea. Viral RNA was extracted from stool samples followed by reverse transcription polymerase chain reaction and nucleotide sequencing of a fragment of the viral protein 1 capsid gene. Multiple sequence alignments were carried out and phylogenetic trees were constructed using the MEGA 6 software.RESULTS Overall, 11.2% of stool samples collected from children aged < 5 years tested positive for No V genogroups Ⅰ(GⅠ) and Ⅱ(GⅡ). GⅡ accounted for 10.6% of the gastroenteritis cases with only five samples being positive for GⅠ(0.7%). The majority of hospitalized children showed symptoms of diarrhea, dehydration, vomiting and fever. Upon sequencing of positive samples and based on their clustering in the phylogenetic tree, 4/5 of GⅠ gastroenteritis cases were designated GⅠ.3 and one case as GⅠ.4. GⅡ.4 was predominantly detected in stool of our study participants(68%). We report a JB-15/KOR/2008 GⅡ.4 Apeldoorn 2008-like variant strain circulating in 2011; this strain was replaced between 2012 and 2013 by a variant sharing homology with the Sydney/NSW0514/2012/AUS GⅡ.4 Sydney 2012 and Sydney 2012/FRA GⅡ.4 strains. We also report the co-circulation of non-GⅡ.4 genotypes among hospitalized children. Our data show that No V gastroenteritis can occur throughout the year with the highest number of cases detected during the hot months.CONCLUSION The majority of No V-associated viral gastroenteritis cases among our participants are attributable to GⅡ.4, which is compatible with results reported worldwide.

猪输血传播病毒2型福建株ORF1基因特征

参照GenBank中登录的猪输血传播病毒2型(Torque Teno Virus genogroup 2,TTV2)ORF1基因序列特征设计特异性引物,采用PCR方法对从福建省某猪场采集的血液基因组DNA中扩增到猪输血传播病毒2型ORF1基因,并对目的片段进行克隆测序。结果表明,所扩增的目的片段编码有TTV2完整的ORF1基因开放阅读框,全长为1 875bp,编码有624个氨基酸。运用生物信息学软件,将获得猪TTV2福建株ORF1基因序列和GenBank中登录的猪TTV2进行分析比较,其和FJ/China/2010/TTV2/2(GenBank登录号JF937656)的同源性高达98.8%,和四川株(GenBank登录号HQ204188)核苷酸同源性最低,仅为83.5%。从遗传进化上看,猪输血传播病毒2型可以分为3个明显的分支亚群,福建分离株在基因1群和基因2群均有分布,推测福建省存在多亚群猪TTV2流行。

实时荧光定量PCR检测GI型诺瓦克样病毒方法的建立

目的建立临床粪便中GI型诺瓦克样病毒的实时荧光定量PCR检测方法。方法针对诺瓦克样病毒GI型保守序列,用序列比对软件设计特异性引物与探针,建立诺瓦克样病毒实时荧光定量PCR检测方法。并用常规RT-PCR和本文建立的实时荧光定量PCR对137份临床腹泻标本进行检测。结果该方法对诺瓦克样病毒检测准确,重复性好,标准曲线的线性范围为102～107拷贝,相关系数为0.9991。并且对临床标本的检出率显著高于普通RT-PCR。结论本研究建立的实时荧光定量PCR方法可用于检测临床腹泻粪便标本中的GI型诺瓦克样病毒,从而有效预防和控制该病毒的传染。

感染性腹泻患者标本中检出GⅡ型诺如病毒

目的为确定引起腹泻的病原体,对腹泻患者标本进行诺如病毒核酸检测。方法应用实时荧光RT-PCR法对5份感染性腹泻患者粪便标本进行GⅠ型和GⅡ诺如病毒RNA检测。结果 5份患者粪便标本中,1份检出GⅡ型诺如病毒核酸。结论大连地区的感染性腹泻病例中存在GⅡ诺如病毒感染。

Monitoring the Hepatitis A Virus in Oyster from Korea

Hepatitis A virus (HAV) causes many cases of oyster- or clam-associated gastroenteritis in various countries. HAV was detected on oyster by RT-PCR in 19.6% (11/56) in Korea. The percentages of HAV-positive samples in 2011 and 2012 were 27.6% and 11.1%, respectively. Phylogenetic analysis revealed that several nucleotide sequences highly similar to those of HAVs isolated in this study. Phylogenetic analysis of the coding regions of the viral protein VP4/VP2 revealed that all amplicons were classi?ed into IA genogroup. It will provide useful data that aids in our understanding circulating HAVs and may contribute to future control.

一步法微滴数字PCR检测生菜中GII型诺如病毒

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。

福州地区腹泻患者诺瓦克样病毒感染的分子流行病学特点

目的 调查研究福州地区腹泻患者诺瓦克样病毒感染的分子流行病学特点。方法 分别应用酶联免疫吸附试验 (ELISA)和逆转录一聚合酶链反应 (RT -PCR) ,对 1 998年 1 0月至 2 0 0 2年 1 1月在我院门诊及住院部收集的 2 88份腹泻患者粪便标本进行轮状病毒和诺瓦克样病毒病原检测。结果 ELISA法检测轮状病毒感染的阳性率为 54 2 % ,发病年龄主要是 1月～ 3岁的婴幼儿 ,发病季节集中在冬春季 ,高峰为 1 1和 1 2月份。RT -PCR检测显示诺瓦克样病毒基因组Ⅰ的阳性率为 1 1 1 % ,主要是 7岁以上儿童及成人 ;诺瓦克样病毒基因组Ⅱ的阳性率为 2 8 8% ,主要是 6月～ 3岁婴幼儿 ;诺瓦克样病毒感染均无明显季节特异性。结论 在福州地区诺瓦克样病毒是仅次于轮状病毒的又一常见腹泻病原 ,其中婴幼儿腹泻以诺瓦克样病毒基因组Ⅱ为主。

实时逆转录环介导等温扩增法快速检测GI型诺如病毒

目的建立实时逆转录环介导等温扩增法(reverse transcription loop-mediated isothermal amplification,RT-LAMP)快速检测GI型诺如病毒的分析方法。方法通过反应引物的筛选、检测温度的优化、灵敏度和特异性研究、样品检测应用等,进行GI型诺如病毒实时RT-LAPM法检测关键技术的研究。结果针对GI型诺如病毒特异性基因,通过设计筛选出最佳实时RT-LAMP引物组,并使用该组引物特异、灵敏、快速地检测到GI型诺如病毒。最佳检测温度为65℃,最小检测限为22.6拷贝/µL,且只对GI型诺如病毒靶基因特异,检测应用结果可靠。结论本研究建立了GI型诺如病毒的实时RT-LAMP检测方法,可用于GI型诺如病毒的样品检测。