诺如病毒荧光定量检测方法的建立及氯灭活诺如病毒消毒规律研究
发布时间：2018-08-06 14:40
【摘要】：研究目的： 诺如病毒（Norovirus，NoV）是一种引起世界范围内非细菌性急性胃肠炎的主要病毒，是杯状病毒家族的主要成员，能够引起急性腹泻、呕吐的发生，重者可因脱水死亡或自身免疫缺陷导致严重的并发症危及生命。从二十世纪九十年代至今，NoV GII.4已经逐渐成为了世界范围内的优势流行株，并且造成了至少四次全球性的疾病流行。NoV主要通过粪口途径传播，食用被病毒污染的食物、人与人的接触、食用牡蛎、饮水都可以传播该病毒，其次通过呼吸道吸入飞扬的呕吐物微粒也可能是一种传播途径。诺如病毒在环境中高度稳定，但是对人却极易感染，因此也是污染饮用水导致腹泻疾病流行的重要病因。上个世纪七十年代，通过电镜发现了NoV，目前，由于缺乏组织细胞培养系统，对于NoV的消毒机理的研究通常采用替代病毒来进行，例如：鼠NoV（murine norovirus，MNV）、猫杯状病毒（feline calicivirus，FeCV)和犬杯状病毒（canine calicivirus，CaCV)。因此，调查研究我国NoV的流行现况，建立快速、准确的检测方法，为制定相应的控制策略和措施提供依据；对该病毒进行消毒研究，可以更好地提供污水消毒指导方案，进一步保证在污水净化过程中的细菌学和病毒学安全指标的达成。因此，本课题通过RT-PCR方法研究2009年10~11月天津市儿童医院的腹泻婴幼儿NoV的流行情况，建立和评价NoV基因II型的荧光定量RT-qPCR检测方法，采用氯对水中NoV的消毒进行初步研究，为污水中NoV的污染检测、消毒控制和突发公共卫生事件的快速检测提供参考方法和依据。 研究方法： 收集天津市2009年10~11月儿童医院门诊部和住院部急性腹泻患儿粪便标本319例，其中男198例、女121例，最小年龄为出生后两天，最大为9岁。通过RT-PCR方法确定NoV核酸阳性的标本，将PCR扩增样本交由公司纯化测序，样本序列结果经过编辑与Genbank中公布的基因进行Blast序列比较，从而判断样本的基因型。根据文献报道，从Genbank下载NoV各型参考株序列。应用Bioedit软件进行核苷酸多序列分析比对，利用MEGA4.1软件邻位相连法绘制系统发生树。采用统计学方法分析样本的人口学资料和临床资料。 采用RT-PCR检测NoV GII强阳性的样本，PCR产物纯化后克隆转化，蓝白斑阳性克隆筛选，构建质粒，，并转录合成copy RNA(cRNA)作为标准品，建立和优化了NoV基因II型荧光定量RT-qPCR方法和反应体系，制作标准曲线，评价该反应体系的灵敏度、特异性、重复性，并进行临床粪便样本的检测评价。 以NoV为研究对象，采用不同浓度的氯（1mg/L、2mg/L、3mg/L、5mg/L、10mg/L）消毒剂处理水中的NoV，使用三对引物对NoV的灭活情况进行评价，其中自行设计分别针对NoV的5’端、3’端的引物，以及ORF1-ORF2结合区的引物参照文献（见第一章）。PH7.2，室温下，不同时间点取样，通过RT-PCR探索氯对NoV基因损伤位点，采用荧光定量PCR方法进一步检测氯消毒过程中NoV的核酸减少量。初步研究氯对NoV的灭活情况。研究结果： 一、2009年10~11月天津市儿童医院腹泻患儿粪便标本中NoV的检出情况 经过RT-PCR检测的319例标本中，共检出60例NoV阳性。RT-PCR检测表明，有59例为NoV GII型，有1例为NoV GI型，构成比分别为98.3%和1.7%。 24例NoV阳性标本PCR产物纯化和测序，测序结果利用bioedit编辑后提交Genbank进行BLAST比对发现，1例是NoV GI型，其余的23例均为NoVGII型。 多序列同源性比对发现，测序毒株17株与荷兰的Nijmegen115参考株、Lincoln House参考株、Beijing151株和Terneuzen70株序列同源性为71.7～99.3%，与Lincoln House参考株同源性高达98.0～99.3%。15例GII-4型中13例是2006b变异株；2例是GII-3型。系统发生树分析显示，2009年天津市腹泻患儿NoV以NoV GII/2006b毒株为主要的流行株，这与我国其他地方目前的研究相似，这表明，目前我国的流行优势株仍然是NoV GII/2006b毒株。 不同年龄组NoV阳性的病例分布情况分析表明，60例感染患儿中，0～1岁年龄组的患儿感染NoV35例，1～2岁患儿感染NoV16例，随着年龄的增大，腹泻患儿减少，感染NoV的儿童也较少。婴幼儿NoV的感染主要是集中于0～2岁人群。 本研究中门诊腹泻患儿NoV的阳性率是26.75%（41/157），而住院部腹泻患儿NoV的阳性率是11.11%(18/162)，统计学分析二者差别有统计学意义，说明该病发病急，自限性，恢复快，大多数病人选择门诊就医。 二、NoV基因II型荧光定量RT-PCR检测方法的建立和评估 NoV GII荧光定量RT-PCR扩增所使用的引物进行普通RT-PCR反应，2%的琼脂糖凝胶电泳显示，在98bp处有特异性条带，没有其他非特异性扩增。该引物和探针荧光定量RT-PCR检测荧光扩增曲线呈典型的光滑的S型曲线。 构建的质粒DNA的纯度好，浓度高，经过体外转录纯化得到cRNA，cRNA的OD值位于1.7~2.0中间，纯度较好，浓度均值为321.83μg/ml。标准曲线的斜率是-3.41，截距是49.79，相关系数（R2）是0.998。 此方法经过灵敏度检测，结果显示敏感性好，最低可以检出102拷贝数/μl的已知浓度RNA标准品样本。 此方法能够特异地检出NoV基因II型，与NoV GI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。 针对标准品的批内试验的Ct值变异系数（CV）分别为1.60%、0.70%，批间试验的变异系数（CV值）分别为0.40%、0.40%，均在5%以下，说明该体系稳定，重复性良好。 三、氯对水中NoV的消毒研究 RT-PCR结果显示，NV3R/NV3F引物对扩增条带最先消失；其次，随着氯的消毒剂量增大，COG2F/G2-SKR引物对扩增条带消失；而后，当氯的消毒剂量从5mg/L到10mg/L时，NV5R/NV5F引物对扩增条带消失。由此，我们认为，在三个不同的扩增中，消毒剂液氯可能优先损伤了NoV核酸的3’端，之后是ORF1与ORF2的结合区，而后损伤了5’端。 PH7.2，室温下，浓度为1mg/L的氯作用下，基于引物COG2F/G2-SKR的荧光定量PCR检测显示，NoV的减少率在20min达到2.16log10。随着氯浓度的增加，在氯浓度5mg/L的时候NoV的减少率在5min可达到2.24log10，在20min达到3.01log10。 结论： 1、2009年10~11月天津市儿童医院腹泻患儿中存在NoV所致的病毒性腹泻，并且NoV是导致腹泻的主要病原体；腹泻患儿存在不同基因型别的NoV感染,NoV GII-4的2006b变异株是主要的流行优势株。 2、我们建立和优化了荧光定量RT-PCR检测NoV GII型的方法，该方法稳定、灵敏性、特异性和重复性良好，可用于突发公共卫生事件的快速检测。 3、在NoV核酸的3’端、ORF1和ORF2的结合区和5’端三个不同的区域中，消毒剂液氯可能先损伤NoV的3’端，之后是ORF1和ORF2的结合区，随着氯的浓度增大时，损伤了NoV的5’端。随着氯使用浓度的增加， NoV的核酸减少率也在增加。
[Abstract]:The purpose of the study is:
Norovirus (NoV) is a major virus that causes non bacterial acute gastroenteritis in the world. It is the main member of the family of the goblet virus. It can cause acute diarrhea, vomiting, and the serious death of the heavy people due to dehydration death or its own immune deficiency. From 1990s to now, NoV G II.4 has gradually become the world's dominant epidemic strain, and has caused at least four global disease epidemics that are spread mainly through the mouth of the.NoV, food contaminated by the virus, human contact, oysters, and drinking water that can spread the virus, and it may also be inhaled through the respiratory tract in the respiratory tract. It is a way of transmission. The virus is highly stable in the environment, but it is very easy to infect people. Therefore, it is also an important cause of the epidemic of diarrhea caused by drinking water. In the 70s of last century, NoV was discovered by electron microscope. At present, the study of the mechanism of NoV disinfection is usually replaced by the lack of tissue cell culture system. Virus, such as mouse NoV (murine norovirus, MNV), cat goblet virus (feline calicivirus, FeCV) and canine goblet virus (canine calicivirus, CaCV). Therefore, investigation and study of the prevalence of NoV in China, establish a rapid and accurate detection method, to provide the basis for the corresponding control strategies and measures; the virus is sterilized and studied. Through the RT-PCR method, the epidemic of NoV in the diarrhoea of Tianjin Children's Hospital in the 2009 10~11 month was studied, and the fluorescence quantitative RT-qPCR of the NoV gene II type was established and evaluated. A preliminary study on the disinfection of NoV in water was carried out by the method of chlorine, which provided reference methods and basis for the detection of pollution of NoV in sewage, the control of disinfection and the rapid detection of public health emergencies.
Research methods:
319 Cases of stool specimens of children with acute diarrhea in the 10~11 month children's Hospital of Tianjin in 2009 were collected, including 198 males and 121 females. The minimum age was two days after birth and the maximum was 9 years. The samples of NoV nucleic acid positive were determined by the RT-PCR method, and the PCR amplification samples were purified and sequenced by the company, and the sample sequence results were edited and Gen The genes published in bank are compared with the Blast sequence to determine the genotype of the samples. According to the literature, the sequence of NoV reference strains are downloaded from Genbank. Bioedit software is used to compare nucleotide multiple sequence analysis and to draw the phylogenetic tree by the MEGA4.1 software adjacent to the site. Statistical method is used to analyze the demographic characteristics of the samples. Materials and clinical data.
The samples of strong positive NoV GII were detected by RT-PCR, the PCR products were purified and transformed, the positive clones of blue leukoplakia were screened, the plasmid was constructed, and copy RNA (cRNA) was transcribed and synthesized as the standard product. The II fluorescent quantitative RT-qPCR method and reaction system of NoV gene were established and optimized, and the standard curve was made, and the sensitivity and specificity of the reaction system were evaluated. Reproducibility and clinical fecal samples were evaluated.
Taking NoV as the research object, using the disinfectant of different concentrations of chlorine (1mg/L, 2mg/L, 3mg/L, 5mg/L, 10mg/L) to treat NoV in water, the inactivation of NoV with three pairs of primers was evaluated, and the primers for the 5 'end, 3' end of NoV and the.PH7.2 primers in the ORF1-ORF2 binding region (see Chapter 1).PH7.2, at room temperature, were not. At the same time, the NoV gene damage site was explored by RT-PCR, and the fluorescence quantitative PCR method was used to further detect the reduction of NoV in the chlorination process. The inactivation of chlorine to NoV was preliminarily studied. The results of the study were as follows:
1. The detection of NoV in stool specimens of children with diarrhea in Tianjin Children's Hospital in 10~11 2009.
Of the 319 specimens examined by RT-PCR, 60 cases of NoV positive.RT-PCR detection showed that 59 cases were NoV GII and 1 were NoV GI, and the constituent ratio was 98.3% and 1.7%., respectively.
The PCR products of 24 cases of NoV positive specimens were purified and sequenced. The results were analyzed by bioedit editing and BLAST comparison. 1 cases were NoV GI and the other 23 were NoVGII type.
Multiple sequence homology comparison found that the sequence homology of 17 strains of sequenced strains and the Nijmegen115 reference strain of Holland, Lincoln House reference strain, Beijing151 strain and Terneuzen70 strain were 71.7 to 99.3%, and the homology of Lincoln House reference strains reached 98 to 99.3%.15 cases, 13 cases were 2006b variant, and 2 cases were GII-3. In 2009, NoV of children with diarrhea in Tianjin city was the main strain of NoV GII/2006b strain, which was similar to the present research in other parts of China. This shows that the prevailing dominant strain in our country is still NoV GII/2006b strain.
Analysis of the distribution of NoV positive cases in different age groups showed that in 60 cases of infected children, the children of 0~1 years old were infected with NoV35, and 1~2 year old children infected NoV16 cases. With the increase of age, the children with diarrhea decreased and the children infected with NoV were also less. The infection of infants with NoV was concentrated on the population of 0~2 years old.
The positive rate of NoV in children with diarrhea in this study was 26.75% (41/157), while the positive rate of NoV in children with diarrhea in the inpatient department was 11.11% (18/162). The statistical analysis of the two differences was statistically significant, indicating that the disease was urgent, self limiting, fast recovery, and most of the patients chose outpatient medical treatment.
Two, the establishment and evaluation of NoV gene II type fluorescence quantitative RT-PCR assay.
The primers used by NoV GII fluorescence quantitative RT-PCR amplification were used for general RT-PCR reaction. 2% agarose gel electrophoresis showed that there were specific bands at 98bp and no other non specific amplification. The primer and probe fluorescence quantitative RT-PCR showed a typical smooth S curve of the fluorescence amplification curve.
The purity of the plasmid DNA was good, the concentration was high, cRNA was purified by transcription in vitro. The OD value of cRNA was in the middle of 1.7~2.0. The purity of the plasmid was better. The slope of the mean concentration of 321.83 mu g/ml. was -3.41, the intercept was 49.79, and the correlation coefficient (R2) was 0.998..
The sensitivity test showed that the method was sensitive and could detect 102 copies/ml of known RNA standard samples at the lowest level.
This method can detect NoV gene II in a special way, without cross reaction with NoV GI type, and no cross reaction with Coxsackie virus B group, poliovirus, enterovirus, stelllike virus, hepatitis A virus, otavirus and rotavirus.
The variation coefficient of Ct value (CV) for the test of standard products was 1.60% and 0.70% respectively. The variation coefficient (CV value) of interbatch test was 0.40% and 0.40% respectively, which were all below 5%, indicating that the system was stable and reproducible.
Three, study on the disinfection of NoV in water by chlorine
RT-PCR results showed that NV3R/NV3F primers were the first to disappear. Secondly, as the dosage of chlorine disinfectant increased, COG2F/G2-SKR primers disappeared. Then, when the dosage of chlorine was from 5mg/L to 10mg/L, NV5R/NV5F primers disappeared. Therefore, we think that the disinfectant liquid chlorine may be in three different amplification. The 3 'terminal of NoV nucleic acid was first damaged, followed by the binding area between ORF1 and ORF2, and then the 5' terminal was damaged.
PH7.2, under the action of 1mg/L at room temperature, the fluorescence quantitative PCR detection based on primer COG2F/G2-SKR showed that the reduction rate of NoV reached 2.16log10. with the increase of chlorine concentration, and the decrease rate of NoV at 5min reached 2.24log10 when the concentration of chlorine was 5mg/L.
Conclusion:
The viral diarrhea caused by NoV in children with diarrhea in Tianjin Children's Hospital in 12009 10~11 months was found, and NoV was the main pathogen causing diarrhea; the children with diarrhea had NoV infection of different genotypes, and the 2006b variant of NoV GII-4 was the main epidemic dominant strain.
2, we established and optimized the method of detecting NoV GII by fluorescence quantitative RT-PCR. This method is stable, sensitive, specific and reproducible, and can be used for rapid detection of public health emergencies.
3, at the 3 'end of NoV nucleic acid, in the binding area of ORF1 and ORF2 and in three different regions of the 5' end, the disinfectant liquid chlorine may first damage the 3 'end of the NoV, and then the binding area of ORF1 and ORF2. As the concentration of chlorine increases, the 5' end of NoV is damaged. As the concentration of chlorine increases, the nucleic acid reduction rate of NoV is also increasing.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5
本文编号：2168051
[Abstract]:The purpose of the study is:
Norovirus (NoV) is a major virus that causes non bacterial acute gastroenteritis in the world. It is the main member of the family of the goblet virus. It can cause acute diarrhea, vomiting, and the serious death of the heavy people due to dehydration death or its own immune deficiency. From 1990s to now, NoV G II.4 has gradually become the world's dominant epidemic strain, and has caused at least four global disease epidemics that are spread mainly through the mouth of the.NoV, food contaminated by the virus, human contact, oysters, and drinking water that can spread the virus, and it may also be inhaled through the respiratory tract in the respiratory tract. It is a way of transmission. The virus is highly stable in the environment, but it is very easy to infect people. Therefore, it is also an important cause of the epidemic of diarrhea caused by drinking water. In the 70s of last century, NoV was discovered by electron microscope. At present, the study of the mechanism of NoV disinfection is usually replaced by the lack of tissue cell culture system. Virus, such as mouse NoV (murine norovirus, MNV), cat goblet virus (feline calicivirus, FeCV) and canine goblet virus (canine calicivirus, CaCV). Therefore, investigation and study of the prevalence of NoV in China, establish a rapid and accurate detection method, to provide the basis for the corresponding control strategies and measures; the virus is sterilized and studied. Through the RT-PCR method, the epidemic of NoV in the diarrhoea of Tianjin Children's Hospital in the 2009 10~11 month was studied, and the fluorescence quantitative RT-qPCR of the NoV gene II type was established and evaluated. A preliminary study on the disinfection of NoV in water was carried out by the method of chlorine, which provided reference methods and basis for the detection of pollution of NoV in sewage, the control of disinfection and the rapid detection of public health emergencies.
Research methods:
319 Cases of stool specimens of children with acute diarrhea in the 10~11 month children's Hospital of Tianjin in 2009 were collected, including 198 males and 121 females. The minimum age was two days after birth and the maximum was 9 years. The samples of NoV nucleic acid positive were determined by the RT-PCR method, and the PCR amplification samples were purified and sequenced by the company, and the sample sequence results were edited and Gen The genes published in bank are compared with the Blast sequence to determine the genotype of the samples. According to the literature, the sequence of NoV reference strains are downloaded from Genbank. Bioedit software is used to compare nucleotide multiple sequence analysis and to draw the phylogenetic tree by the MEGA4.1 software adjacent to the site. Statistical method is used to analyze the demographic characteristics of the samples. Materials and clinical data.
The samples of strong positive NoV GII were detected by RT-PCR, the PCR products were purified and transformed, the positive clones of blue leukoplakia were screened, the plasmid was constructed, and copy RNA (cRNA) was transcribed and synthesized as the standard product. The II fluorescent quantitative RT-qPCR method and reaction system of NoV gene were established and optimized, and the standard curve was made, and the sensitivity and specificity of the reaction system were evaluated. Reproducibility and clinical fecal samples were evaluated.
Taking NoV as the research object, using the disinfectant of different concentrations of chlorine (1mg/L, 2mg/L, 3mg/L, 5mg/L, 10mg/L) to treat NoV in water, the inactivation of NoV with three pairs of primers was evaluated, and the primers for the 5 'end, 3' end of NoV and the.PH7.2 primers in the ORF1-ORF2 binding region (see Chapter 1).PH7.2, at room temperature, were not. At the same time, the NoV gene damage site was explored by RT-PCR, and the fluorescence quantitative PCR method was used to further detect the reduction of NoV in the chlorination process. The inactivation of chlorine to NoV was preliminarily studied. The results of the study were as follows:
1. The detection of NoV in stool specimens of children with diarrhea in Tianjin Children's Hospital in 10~11 2009.
Of the 319 specimens examined by RT-PCR, 60 cases of NoV positive.RT-PCR detection showed that 59 cases were NoV GII and 1 were NoV GI, and the constituent ratio was 98.3% and 1.7%., respectively.
The PCR products of 24 cases of NoV positive specimens were purified and sequenced. The results were analyzed by bioedit editing and BLAST comparison. 1 cases were NoV GI and the other 23 were NoVGII type.
Multiple sequence homology comparison found that the sequence homology of 17 strains of sequenced strains and the Nijmegen115 reference strain of Holland, Lincoln House reference strain, Beijing151 strain and Terneuzen70 strain were 71.7 to 99.3%, and the homology of Lincoln House reference strains reached 98 to 99.3%.15 cases, 13 cases were 2006b variant, and 2 cases were GII-3. In 2009, NoV of children with diarrhea in Tianjin city was the main strain of NoV GII/2006b strain, which was similar to the present research in other parts of China. This shows that the prevailing dominant strain in our country is still NoV GII/2006b strain.
Analysis of the distribution of NoV positive cases in different age groups showed that in 60 cases of infected children, the children of 0~1 years old were infected with NoV35, and 1~2 year old children infected NoV16 cases. With the increase of age, the children with diarrhea decreased and the children infected with NoV were also less. The infection of infants with NoV was concentrated on the population of 0~2 years old.
The positive rate of NoV in children with diarrhea in this study was 26.75% (41/157), while the positive rate of NoV in children with diarrhea in the inpatient department was 11.11% (18/162). The statistical analysis of the two differences was statistically significant, indicating that the disease was urgent, self limiting, fast recovery, and most of the patients chose outpatient medical treatment.
Two, the establishment and evaluation of NoV gene II type fluorescence quantitative RT-PCR assay.
The primers used by NoV GII fluorescence quantitative RT-PCR amplification were used for general RT-PCR reaction. 2% agarose gel electrophoresis showed that there were specific bands at 98bp and no other non specific amplification. The primer and probe fluorescence quantitative RT-PCR showed a typical smooth S curve of the fluorescence amplification curve.
The purity of the plasmid DNA was good, the concentration was high, cRNA was purified by transcription in vitro. The OD value of cRNA was in the middle of 1.7~2.0. The purity of the plasmid was better. The slope of the mean concentration of 321.83 mu g/ml. was -3.41, the intercept was 49.79, and the correlation coefficient (R2) was 0.998..
The sensitivity test showed that the method was sensitive and could detect 102 copies/ml of known RNA standard samples at the lowest level.
This method can detect NoV gene II in a special way, without cross reaction with NoV GI type, and no cross reaction with Coxsackie virus B group, poliovirus, enterovirus, stelllike virus, hepatitis A virus, otavirus and rotavirus.
The variation coefficient of Ct value (CV) for the test of standard products was 1.60% and 0.70% respectively. The variation coefficient (CV value) of interbatch test was 0.40% and 0.40% respectively, which were all below 5%, indicating that the system was stable and reproducible.
Three, study on the disinfection of NoV in water by chlorine
RT-PCR results showed that NV3R/NV3F primers were the first to disappear. Secondly, as the dosage of chlorine disinfectant increased, COG2F/G2-SKR primers disappeared. Then, when the dosage of chlorine was from 5mg/L to 10mg/L, NV5R/NV5F primers disappeared. Therefore, we think that the disinfectant liquid chlorine may be in three different amplification. The 3 'terminal of NoV nucleic acid was first damaged, followed by the binding area between ORF1 and ORF2, and then the 5' terminal was damaged.
PH7.2, under the action of 1mg/L at room temperature, the fluorescence quantitative PCR detection based on primer COG2F/G2-SKR showed that the reduction rate of NoV reached 2.16log10. with the increase of chlorine concentration, and the decrease rate of NoV at 5min reached 2.24log10 when the concentration of chlorine was 5mg/L.
Conclusion:
The viral diarrhea caused by NoV in children with diarrhea in Tianjin Children's Hospital in 12009 10~11 months was found, and NoV was the main pathogen causing diarrhea; the children with diarrhea had NoV infection of different genotypes, and the 2006b variant of NoV GII-4 was the main epidemic dominant strain.
2, we established and optimized the method of detecting NoV GII by fluorescence quantitative RT-PCR. This method is stable, sensitive, specific and reproducible, and can be used for rapid detection of public health emergencies.
3, at the 3 'end of NoV nucleic acid, in the binding area of ORF1 and ORF2 and in three different regions of the 5' end, the disinfectant liquid chlorine may first damage the 3 'end of the NoV, and then the binding area of ORF1 and ORF2. As the concentration of chlorine increases, the 5' end of NoV is damaged. As the concentration of chlorine increases, the nucleic acid reduction rate of NoV is also increasing.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5
【参考文献】
相关期刊论文 前10条
1 陈冬梅,张又,钱渊,Jason Jiang;我国婴幼儿中存在不同基因型杯状病毒的感染[J];病毒学报;2001年03期
2 方肇寅，温乐英，晋圣瑾，赵章华;在我国腹泻患儿中发现诺瓦克样病霉感染[J];病毒学报;1995年03期
3 靖宇,钱渊,王洛平;北京地区人群诺瓦克样病毒血清抗体水平调查[J];病毒学报;1998年04期
4 张颖;郑辉;林杨;张伟;李英军;马雯;马晓燕;马玉青;;诺瓦克样病毒及其检测方法[J];现代食品科技;2006年04期
5 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年08期
6 欧阳雅博;马慧;金敏;陈照立;王新为;王景峰;谌志强;邱志刚;彭林;李君文;;天津市婴幼儿病毒性腹泻诺如病毒检测[J];中国公共卫生;2010年06期
7 靖宇,钱渊,吴立平,马建瑞;太原市部分人群诺瓦克样病毒血清抗体水平的调查[J];中华儿科杂志;1999年09期
8 吕红霞,方肇寅,谢华萍,唐景裕,胡海宽,郑丽舒,叶青,章青;河北省卢龙县1999～2001年婴幼儿杯状病毒腹泻流行病学研究[J];中华流行病学杂志;2003年12期
9 高志勇;吕虹;黄芳;吴晓娜;严寒秋;刘园;刘桂荣;李洁;窦相锋;王全意;庄辉;;90例病毒性胃肠炎患者粪便中诺如病毒检出情况分析[J];中国病原生物学杂志;2008年08期
10 王作唈;韩悦;安淑一;郭军巧;;诺如病毒RT-PCR检测及进化树分析[J];中国卫生检验杂志;2008年05期
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