人感染高致病性禽流感病毒H5N1核酸检测方法的建立及应用

目的 建立人感染高致病性禽流感病毒H5N1的核酸检测方法,用于人感染高致病性禽流感病毒疑似病例临床标本的检测.方法 针对甲型流感病毒保守基因M设计RT-PCR和real-time PCR引物检测是否为甲型流感病毒,同时针对H5N1禽流感病毒设计针对H5和N1基因的特异性RT-PCR和real-time PCR引物作亚型检测,建立禽流感H5N1病毒RT-PCR和real-time PCR检测方法.结果 本研究建立的RT-PCR和real-time PCR方法可以特异性地检测H5N1病毒,并且与人流感病毒H1、H3没有交叉反应.RT-PCR检测方法灵敏度可到1TCID50,real-time PCR灵敏度可达0.01TCID50.利用上述方法检测人感染高致病性禽流感病毒H5N1疑似病例临床标本,从42例不明原因肺炎病例中检测出阳性标本13例.结论 本研究建立的RT-PCR和real-time PCR方法可以用于人感染高致病性禽流感病毒H5N1临床标本的实验室检测.     

草莓中甲型肝炎病毒检测方法的比较

目的对草莓中甲型肝炎病毒(hepatitis A virus, HAV)检测的两种检测方法进行优化比较, 以选出最佳的检测方法, 用于草莓中甲肝病毒的检测。方法向已知阴性冷冻草莓标本表面接种不同浓度的HAV, 优化碱性洗脱-PEG浓缩法中的牛肉浸出粉的浓度以及选择最适核酸提取试剂盒, 优化直接裂解法中的最适裂解缓冲液体积, 对草莓标本进行处理, 采用实时荧光定量RT-PCR检测回收的病毒量, 应用SPSS26.0对数据进行统计分析, 并将优化后的两种方法用于实际标本的检测。结果优化后的碱性洗脱-PEG浓缩法的牛肉浸出粉浓度选择3%, 病毒核酸提取试剂盒选择试剂盒B。优化后的直接裂解法的裂解缓冲液体积选择6 ml。对碱性洗脱-PEG浓缩法、直接裂解法在添加HAV不同浓度水平进行比较, 两种方法的HAV病毒回收率分别为21.50±1.06%、5.82±0.01%, 结果表明差异具有统计学意义。同时对四个地区共60份草莓标本进行检测, 结果均为阴性。结论优化后的碱性洗脱-PEG浓缩法灵敏度更高, 更适用于草莓标本中HAV的检测。     

河北石家庄2005-2007年甲肝病毒流行株基因分型研究

目的 了解石家庄地区甲型肝炎病毒(HAV)流行株基因型特征,为HAV溯源研究打下基础.方法 收集了2005-2007年石家庄地区部分甲肝患者急性期血清标本,用HAV结构-非结构区VP1-2A基因引物,经核酸提取,RT-PeR,序列测定,对HAV进行基因分型分析.结果 石家庄地区2005-2007年HAV流行株VP1-2A区核苷酸序列同源性为95%～100%,都属于Ⅰ A亚型;该区氨基酸序列几乎相同.结论 石家庄地区存在有多株甲肝病毒流行株,同一株甲肝病毒可以存在不同地区,同一地区可以检测到相同或不相同的HAV毒株.为今后进一步开展甲肝病毒分子流行病学研究及有效控制HAV流行提供了理论和技术支撑.     

甲肝病毒特异性TaqMan荧光定量PCR法检测甲肝病毒核酸的研究

目的 建立甲肝病毒(HAY)特异性TaqMan荧光定量PCR检测方法,并对血清等样品中的HAV进行检测.方法 根据参考文献,选取HAV基因组保守区5'-NCR区引物和探针,建立特异性强、敏感度高、稳定性好的TaqMan HAV Real-time RT-PCR检测体系并应用于甲肝患者血清等病毒核酸的检测.结果 本研究建立的方法能够特异检测出样品中的甲肝病毒,其灵敏度介于每反应0.1CCID50至0.01CCID50之间.同一样本重复检测3次,批内样本Ct值的变异系数最大2.0％,批间样本Ct值的变异系数最大2.6％.急性期甲型肝炎患者血清中甲肝病毒为5.18×102～4.93×107拷贝/ml.结论 本实验建立的HAV Real-time RT-PCR检测方法具有特异、灵敏、快速等优点,可成功应用于临床样本等甲肝病毒的病原学检测.     

双重荧光定量RT—PCR在快速检测A、B型流感病毒上的应用

目的 建立双重荧光定量RT-PCR技术同时快速检测A、B型流感病毒,并应用于临床样本的检测.方法 在A型流感病毒M基因和B型流感病毒HA基因的保守区序列分别设计特异性引物和Taqman探针,建立优化双重荧光RT-PCR反应体系,评价所建双重RT-PCR反应体系的特异性、敏感性和稳定性,并应用于疑似流感含漱液标本检测.结果 该方法对A、B型流感病毒检测具有高度特异性,检出限分别为0.1TCID50和0.01TCID50,具有较好的稳定性.可从疑似流感患者含漱液中直接检测到流感病毒核酸.结论 本研究建立的双重荧光定量RT-PCR可以同时准确分型A、B型流感病毒,灵敏度高,稳定性好,是一种快速检测流感病毒的新方法.     

新疆和田2006年甲肝病毒流行株基因分型研究

目的 了解2006年新疆和田甲型肝炎病毒(HAV)流行株基因型特征,为HAV溯源研究打下基础.方法 收集了新疆和田部分甲肝病人血清标本,用HAV结构.非结构区基因VP1-2A引物,经核酸提取,RT-PCR,序列测定,对HAV进行基因分型研究.结果 新疆和田VP1-2A区HAV核苷酸序列变异为0%～3.9%,分为不同基因簇,但都属1A亚型;VP1-2A区氨基酸序列只有0～2个差异.与已发表的2005年新疆伊犁部分甲肝病毒流行株基因有相同序列或同源性较高.结论 和田有多株甲肝病毒存在,本研究流行可能有多个传染源,多个传播链,在人群免疫水平较低时,引起甲肝流行.结果 表明HAV分子流行病学方法在HAV流行株遗传变异及溯源研究中,以及控制HAV流行中具有重要作用.     

多重RT-PCR方法检测甲型流感病毒

目的建立一种快速、敏感、特异的多重RT-PCR,同时检测甲型流感病毒中的3个分型:甲型H1N1流感病毒,季节性H1N1流感病毒,季节性H3N2流感病毒,并将此方法应用到实验室流感病毒核酸检测技术中。方法利用甲型流感病毒3个分型病毒的引物,在同一个RT-PCR反应体系中,对疑似流感咽拭子标本进行检测。结果多重RT-PCR对甲型流感病毒中分型病毒有较高的灵敏度和特异性,可直接从疑似流感标本中同时进行甲型流感病毒分型检测。结论此实验中采用的多重RT-PCR具有与常规RT-PCR一样的特异性和敏感度,而且比普通RT-PCR和病毒分离法更快速,也更简便。     

福建省首株黄热病毒株的分离鉴定和全基因组序列分析

目的:从福建省2016年输入性黄热病病例标本中分离黄热病毒(YFV)并分析其生物学特征。方法:将16份黄热病毒核酸阳性血清、尿液、唾液样品分别接种C6/36细胞,YFV实时荧光RT-PCR法鉴定检测培养物中特异性病毒核酸,通过高通量测序获得病毒全基因序列并绘制系统进化树。结果:从1例患者发病后3天的血清样品分离到一株病...     

甲型肝炎病毒四种核酸检测方法的比较

目的:比较甲型肝炎病毒(hepatitis A virus,HAV)四种核酸检测方法。方法:采用A、B、C实时荧光定量RT-PCR(RT-qPCR)及D微滴芯片数字RT-PCR(RT-dPCR)分别对HAV质粒标准品、梯度稀释的HAV疫苗进行灵敏度检测;对相关病毒核酸进行特异性检测;用A、B、C方法对40份人工污染HA...     

中国大陆首例人感染高致病性禽流感病毒(H5N1)病例的实验室诊断

目的 对2005年10月湖南省湘潭市湘潭县发生的一名不明原因肺炎病例进行实验室检测,以确定导致该病例的主要病因.方法 采集病例呼吸道标本以及血清标本,对呼吸道标本利用分子鉴别诊断技术以及RT-PCR和实时荧光定量RT-PCR检测病毒核酸;通过血凝抑制试验以及微量中和试验检测血清中的特异性抗体.结果 该病例所有的呼吸道标本H5N1病毒特异性核酸及病毒分离均为阴性.红细胞凝集抑制及微量中和实验显示,恢复期血清较急性期血清H5N1特异性抗体阳转并且有4倍以上增高.结论 通过实验室检测结果分析,该病例为中国大陆第一例人感染高致病性禽流感病毒（H5N1）实验室确诊病例.     

Exposure to multiple subgenotypes of hepatitis A virus during an outbreak using matched serum and saliva specimens

Matched serum and saliva samples were collected simultaneously from 124 subjects exposed during a hepatitis A virus (HAV) outbreak at a daycare center in Rio de Janeiro, Brazil. All samples were tested for IgM and total anti-HAV antibodies by enzyme immunoassay (EIA). HAV was detected by nested PCR in serum, saliva, and water samples employing primers for the VP1/2A region of the viral RNA; all positive products were then sequenced. The viral load of the matched samples was determined by real-time PCR using the TaqMan system. HAV-RNA was identified by nested PCR in 37.7% of the saliva samples, 29% of the serum samples, and one drinking water sample. The mean HAV viral load was similar in the serum and saliva specimens (10(3) copies/ml). HAV genotypes IA and IB were detected in both specimen types, and the water sample isolate was classified as genotype IB, indicating the existence of more than one source of infection at the daycare center. In six infected patients, a different HAV subgenotype was found in their serum than in their saliva, and this unusual pattern of mixed HAV infection was investigated further by molecular cloning followed by nucleotide sequencing. All clones derived from the saliva samples belonged to subgenotype IB and shared 96.5-100% identity. However, clones derived from their corresponding serum sample belonged to subgenotype IA and shared 90.5-100% identity. This study showed the important role that non-invasive saliva samples can play in the molecular epidemiological analysis of a hepatitis A outbreak.     

Comparison between serum and saliva for the detection of hepatitis A virus RNA

Due to the ease of collection, oral fluid is being investigated as an alternative to serum for diagnostic and epidemiological purposes. However, for prospective studies involving hepatitis A virus (HAV) RNA detection, a standard methodology must be developed. In the present study, nested RT-PCR and real-time PCR were optimized and evaluated for HAV detection and quantification, using oral fluid from healthy volunteers (n=20) and paired serum/oral fluid samples from individuals involved in a hepatitis A outbreak (n=78). Using nested RT-PCR, HAV RNA was detected in 50% of oral fluid and in 42% of serum samples from acute cases, as well as in 12% of all samples from cases without IgM and total anti-HAV. Using real-time PCR, HAV RNA was detected in 61% of oral fluid and in 71% of serum samples from acute cases, as well as in 17 and 12%, respectively, from patients without HAV markers. Mean viral loads were 1.7+/-3.24 x 10(3)copies/ml in oral fluid and 2.8+/-6.46 x 10(3)copies/ml in serum. Although nested RT-PCR and real-time PCR both detected HAV RNA in oral fluid, real-time PCR was more sensitive. Oral fluid sample testing could be used as a noninvasive method of detecting HAV RNA during HAV outbreaks.     

Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions

Vegetables can be considered as a vector of transmission for human hepatic and enteric viruses such as hepatitis A virus (HAV) and noroviruses when contaminated by spoiled irrigation water or when prepared by infected food handlers. Recently, outbreaks of HAV have been reported in the USA involving fresh green onions. A viral elution-concentration method was developed for the detection of HAV and norovirus contaminated green onions by RT-PCR. Repeated pipetting/washings of the surface with a pH 9.5 glycine-buffered solution allowed the elution of viruses from the vegetables. Concentration of the viral load was performed by a polyethylene glycol (PEG) precipitation procedure. Viral RNAs were extracted and purified using a combination of Trizol-chloroform and poly(dT) magnetic beads methods. Different sets of primers, including two newly designed primers sets for HAV RT-PCR, were tested in order to achieve the best analytical sensitivity. Using the new primer design, it was possible to detect 10(0) TCID(50%)/25 g of HAV in fresh green onions, while 1 RT-PCRU/25 g was detected for noroviruses GII using previously described primers. This method, based on molecular tools, would be useful for diagnostic laboratories in order to perform viral analyses of such commodities as fresh vegetables in cases of foodborne infections.     

Recovery and detection of enterovirus, hepatitis A virus and Norwalk virus in hardshell clams (Mercenaria mercenaria) by RT-PCR methods

A method for recovery of enteric viruses from hardshell clams (Mercenaria mercenaria) has been developed and evaluated. Seeded 50-g samples of clam tissue homogenates were processed by adsorption elution precipitation, two fluorocarbon extractions and PEG precipitation. Clam concentrates were assayed by infectivity and by RT-PCR after guanidinium isothiocyanate (GIT) extraction and/or an indirect immunomagnetic capture (IC) of the virus using paramagnetic beads. GIT extraction removed PCR inhibitors and allowed a reliable RT-PCR detection of viral RNA. The detection sensitivity of GIT extraction-RT-PCR was < 1 PFU of poliovirus 1, < 10 PFU of HAV and 1-11 PCRU of Norwalk virus. IC was very effective for additional concentration and purification of enteric viruses from clam concentrates removing most RT-PCR inhibitors. The sensitivity of this method was comparable to the GIT extraction and the sample volume tolerance for PCR was increased about 10-fold. Both methods gave similar efficiency for virus detection in samples seeded with low virus levels. The procedure developed in this study is effective for enteric viruses detection in hardshell clams by RT-PCR.