諾如病毒熒光定量檢測(cè)方法的建立及氯滅活諾如病毒消毒規(guī)律研究
發(fā)布時(shí)間：2018-08-06 14:40
【摘要】：研究目的： 諾如病毒（Norovirus，NoV）是一種引起世界范圍內(nèi)非細(xì)菌性急性胃腸炎的主要病毒，是杯狀病毒家族的主要成員，能夠引起急性腹瀉、嘔吐的發(fā)生，重者可因脫水死亡或自身免疫缺陷導(dǎo)致嚴(yán)重的并發(fā)癥危及生命。從二十世紀(jì)九十年代至今，NoV GII.4已經(jīng)逐漸成為了世界范圍內(nèi)的優(yōu)勢(shì)流行株，并且造成了至少四次全球性的疾病流行。NoV主要通過糞口途徑傳播，食用被病毒污染的食物、人與人的接觸、食用牡蠣、飲水都可以傳播該病毒，其次通過呼吸道吸入飛揚(yáng)的嘔吐物微粒也可能是一種傳播途徑。諾如病毒在環(huán)境中高度穩(wěn)定，但是對(duì)人卻極易感染，因此也是污染飲用水導(dǎo)致腹瀉疾病流行的重要病因。上個(gè)世紀(jì)七十年代，通過電鏡發(fā)現(xiàn)了NoV，目前，由于缺乏組織細(xì)胞培養(yǎng)系統(tǒng)，對(duì)于NoV的消毒機(jī)理的研究通常采用替代病毒來進(jìn)行，例如：鼠NoV（murine norovirus，MNV）、貓杯狀病毒（feline calicivirus，F(xiàn)eCV)和犬杯狀病毒（canine calicivirus，CaCV)。因此，調(diào)查研究我國(guó)NoV的流行現(xiàn)況，建立快速、準(zhǔn)確的檢測(cè)方法，為制定相應(yīng)的控制策略和措施提供依據(jù)；對(duì)該病毒進(jìn)行消毒研究，可以更好地提供污水消毒指導(dǎo)方案，進(jìn)一步保證在污水凈化過程中的細(xì)菌學(xué)和病毒學(xué)安全指標(biāo)的達(dá)成。因此，本課題通過RT-PCR方法研究2009年10~11月天津市兒童醫(yī)院的腹瀉嬰幼兒NoV的流行情況，建立和評(píng)價(jià)NoV基因II型的熒光定量RT-qPCR檢測(cè)方法，采用氯對(duì)水中NoV的消毒進(jìn)行初步研究，為污水中NoV的污染檢測(cè)、消毒控制和突發(fā)公共衛(wèi)生事件的快速檢測(cè)提供參考方法和依據(jù)。 研究方法： 收集天津市2009年10~11月兒童醫(yī)院門診部和住院部急性腹瀉患兒糞便標(biāo)本319例，其中男198例、女121例，最小年齡為出生后兩天，最大為9歲。通過RT-PCR方法確定NoV核酸陽(yáng)性的標(biāo)本，將PCR擴(kuò)增樣本交由公司純化測(cè)序，樣本序列結(jié)果經(jīng)過編輯與Genbank中公布的基因進(jìn)行Blast序列比較，從而判斷樣本的基因型。根據(jù)文獻(xiàn)報(bào)道，從Genbank下載NoV各型參考株序列。應(yīng)用Bioedit軟件進(jìn)行核苷酸多序列分析比對(duì)，利用MEGA4.1軟件鄰位相連法繪制系統(tǒng)發(fā)生樹。采用統(tǒng)計(jì)學(xué)方法分析樣本的人口學(xué)資料和臨床資料。 采用RT-PCR檢測(cè)NoV GII強(qiáng)陽(yáng)性的樣本，PCR產(chǎn)物純化后克隆轉(zhuǎn)化，藍(lán)白斑陽(yáng)性克隆篩選，構(gòu)建質(zhì)粒，，并轉(zhuǎn)錄合成copy RNA(cRNA)作為標(biāo)準(zhǔn)品，建立和優(yōu)化了NoV基因II型熒光定量RT-qPCR方法和反應(yīng)體系，制作標(biāo)準(zhǔn)曲線，評(píng)價(jià)該反應(yīng)體系的靈敏度、特異性、重復(fù)性，并進(jìn)行臨床糞便樣本的檢測(cè)評(píng)價(jià)。 以NoV為研究對(duì)象，采用不同濃度的氯（1mg/L、2mg/L、3mg/L、5mg/L、10mg/L）消毒劑處理水中的NoV，使用三對(duì)引物對(duì)NoV的滅活情況進(jìn)行評(píng)價(jià)，其中自行設(shè)計(jì)分別針對(duì)NoV的5’端、3’端的引物，以及ORF1-ORF2結(jié)合區(qū)的引物參照文獻(xiàn)（見第一章）。PH7.2，室溫下，不同時(shí)間點(diǎn)取樣，通過RT-PCR探索氯對(duì)NoV基因損傷位點(diǎn)，采用熒光定量PCR方法進(jìn)一步檢測(cè)氯消毒過程中NoV的核酸減少量。初步研究氯對(duì)NoV的滅活情況。研究結(jié)果： 一、2009年10~11月天津市兒童醫(yī)院腹瀉患兒糞便標(biāo)本中NoV的檢出情況 經(jīng)過RT-PCR檢測(cè)的319例標(biāo)本中，共檢出60例NoV陽(yáng)性。RT-PCR檢測(cè)表明，有59例為NoV GII型，有1例為NoV GI型，構(gòu)成比分別為98.3%和1.7%。 24例NoV陽(yáng)性標(biāo)本PCR產(chǎn)物純化和測(cè)序，測(cè)序結(jié)果利用bioedit編輯后提交Genbank進(jìn)行BLAST比對(duì)發(fā)現(xiàn)，1例是NoV GI型，其余的23例均為NoVGII型。 多序列同源性比對(duì)發(fā)現(xiàn)，測(cè)序毒株17株與荷蘭的Nijmegen115參考株、Lincoln House參考株、Beijing151株和Terneuzen70株序列同源性為71.7～99.3%，與Lincoln House參考株同源性高達(dá)98.0～99.3%。15例GII-4型中13例是2006b變異株；2例是GII-3型。系統(tǒng)發(fā)生樹分析顯示，2009年天津市腹瀉患兒NoV以NoV GII/2006b毒株為主要的流行株，這與我國(guó)其他地方目前的研究相似，這表明，目前我國(guó)的流行優(yōu)勢(shì)株仍然是NoV GII/2006b毒株。 不同年齡組NoV陽(yáng)性的病例分布情況分析表明，60例感染患兒中，0～1歲年齡組的患兒感染NoV35例，1～2歲患兒感染NoV16例，隨著年齡的增大，腹瀉患兒減少，感染NoV的兒童也較少。嬰幼兒NoV的感染主要是集中于0～2歲人群。 本研究中門診腹瀉患兒NoV的陽(yáng)性率是26.75%（41/157），而住院部腹瀉患兒NoV的陽(yáng)性率是11.11%(18/162)，統(tǒng)計(jì)學(xué)分析二者差別有統(tǒng)計(jì)學(xué)意義，說明該病發(fā)病急，自限性，恢復(fù)快，大多數(shù)病人選擇門診就醫(yī)。 二、NoV基因II型熒光定量RT-PCR檢測(cè)方法的建立和評(píng)估 NoV GII熒光定量RT-PCR擴(kuò)增所使用的引物進(jìn)行普通RT-PCR反應(yīng)，2%的瓊脂糖凝膠電泳顯示，在98bp處有特異性條帶，沒有其他非特異性擴(kuò)增。該引物和探針熒光定量RT-PCR檢測(cè)熒光擴(kuò)增曲線呈典型的光滑的S型曲線。 構(gòu)建的質(zhì)粒DNA的純度好，濃度高，經(jīng)過體外轉(zhuǎn)錄純化得到cRNA，cRNA的OD值位于1.7~2.0中間，純度較好，濃度均值為321.83μg/ml。標(biāo)準(zhǔn)曲線的斜率是-3.41，截距是49.79，相關(guān)系數(shù)（R2）是0.998。 此方法經(jīng)過靈敏度檢測(cè)，結(jié)果顯示敏感性好，最低可以檢出102拷貝數(shù)/μl的已知濃度RNA標(biāo)準(zhǔn)品樣本。 此方法能夠特異地檢出NoV基因II型，與NoV GI型無交叉反應(yīng)，與柯薩奇病毒B組、脊髓灰質(zhì)炎病毒、腸道病毒、星狀病毒、甲肝病毒、埃可病毒和輪狀病毒無交叉反應(yīng)。 針對(duì)標(biāo)準(zhǔn)品的批內(nèi)試驗(yàn)的Ct值變異系數(shù)（CV）分別為1.60%、0.70%，批間試驗(yàn)的變異系數(shù)（CV值）分別為0.40%、0.40%，均在5%以下，說明該體系穩(wěn)定，重復(fù)性良好。 三、氯對(duì)水中NoV的消毒研究 RT-PCR結(jié)果顯示，NV3R/NV3F引物對(duì)擴(kuò)增條帶最先消失；其次，隨著氯的消毒劑量增大，COG2F/G2-SKR引物對(duì)擴(kuò)增條帶消失；而后，當(dāng)氯的消毒劑量從5mg/L到10mg/L時(shí)，NV5R/NV5F引物對(duì)擴(kuò)增條帶消失。由此，我們認(rèn)為，在三個(gè)不同的擴(kuò)增中，消毒劑液氯可能優(yōu)先損傷了NoV核酸的3’端，之后是ORF1與ORF2的結(jié)合區(qū)，而后損傷了5’端。 PH7.2，室溫下，濃度為1mg/L的氯作用下，基于引物COG2F/G2-SKR的熒光定量PCR檢測(cè)顯示，NoV的減少率在20min達(dá)到2.16log10。隨著氯濃度的增加，在氯濃度5mg/L的時(shí)候NoV的減少率在5min可達(dá)到2.24log10，在20min達(dá)到3.01log10。 結(jié)論： 1、2009年10~11月天津市兒童醫(yī)院腹瀉患兒中存在NoV所致的病毒性腹瀉，并且NoV是導(dǎo)致腹瀉的主要病原體；腹瀉患兒存在不同基因型別的NoV感染,NoV GII-4的2006b變異株是主要的流行優(yōu)勢(shì)株。 2、我們建立和優(yōu)化了熒光定量RT-PCR檢測(cè)NoV GII型的方法，該方法穩(wěn)定、靈敏性、特異性和重復(fù)性良好，可用于突發(fā)公共衛(wèi)生事件的快速檢測(cè)。 3、在NoV核酸的3’端、ORF1和ORF2的結(jié)合區(qū)和5’端三個(gè)不同的區(qū)域中，消毒劑液氯可能先損傷NoV的3’端，之后是ORF1和ORF2的結(jié)合區(qū)，隨著氯的濃度增大時(shí)，損傷了NoV的5’端。隨著氯使用濃度的增加， NoV的核酸減少率也在增加。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5
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