快速检测流感病毒抗原方法的建立

目的建立有效、简便的胶体金免疫层析方法（G ICA）快速检测流感病毒感染抗原方法。方法分别对1145例急性呼吸道疾病住院患者应用G ICA法检测患者鼻咽部分泌物的流感病毒A、B（FluA、B）抗原,并同时用IFA法作对照,对G ICA法做出评价。对以上患者分别应用鼻、咽拭子法和鼻咽部细导管负压吸引法两种不同方法采集患者鼻咽部分泌物,均经G ICA法快速检测分泌物标本中的Flu A、B抗原,对两种不同采集方法的结果做出评价。结果以上两种检测方法及采集方法所得结果,均有较好的一致性,之间差异无统计学意义（P〉0.05）。结论鼻、咽拭子采集法配合G ICA法是简便、快捷、敏感的检测流感病毒抗原的好方法。     

禽流感病毒特异性NP单克隆抗体的鉴定及禽流感特异性检测方法的建立

目的筛选鉴定禽流感病毒特异性单克隆抗体(单抗)并建立一种适合禽源流感病毒特异性检测的抗原检测方法。方法利用分子进化树分析方法对甲型流感病毒核蛋白(NP)基因进行分类,从禽流感病毒分支和人流感病毒分支上各挑选一个代表性流感病毒株的NP基因进行重组抗原的表达及其单抗的筛选制备,最后应用免疫渗滤技术(A-Dot-ELISA)建立抗原快速检测方法。结果根据分子进化树分析结果确定禽流感H5N1病毒株HK/212/2003和人流感H1N1病毒株CA/04/2009的NP基因进行原核表达,获得高纯度的禽流感病毒NP重组抗原rNP-HK/212和人流感病毒NP重组抗原rNP-CA/04;利用上述两种NP抗原制备出抗rNP-HK/212单抗53株,通过差异筛选获得只对禽流感病毒NP蛋白rNP-HK/212有特异性反应而不与人流感病毒NP反应的3株单抗(1F2,5D2及25A2);免疫荧光方法证实1F2等3株单抗只特异识别禽流感病毒;利用单抗1F2成功建立一种适于禽流感病毒特异性检测的抗原快速检测方法 AIV-Dot-ELISA,该方法对病毒滴度为0.025~0.1HA titer的19个不同亚型的禽流感病毒均能检出,对病毒滴度为5HA titer的8个人流感病毒和2个乙型流感病毒的检测均为阴性。结论本研究成功制备出禽流感病毒NP蛋白特异性单抗,并建立了一种仅特异识别禽流感病毒的抗原快速检测方法 AIV-Dot-ELISA,为快速鉴别禽类流感病毒和人类流感病毒感染提供了一种有用的分析检测工具。     

斑点免疫酶渗滤法快速检测甲型流感病毒的临床应用

目的 明确快速筛查甲型流感病毒的方法.方法 收集2009年11月至2010年3月就诊于广州医学院第一附属医院发热门诊及中医科门诊40例流感样患者的鼻咽分泌物进行病毒检测,男18例,女22例,年龄5～62岁,平均年龄(31.3±13.2)岁.以2009年9月卫生部颁布的qRT-PCR方法检出甲型流感病毒作为诊断标准,比较2种快速病原检测法——斑点免疫酶渗滤法(DIEFA)和胶体金免疫层析法(GICA)对甲型流感病毒的检出率,以确定敏感性好、特异性高的方法为甲型流感病毒快速筛查法.结果 40例标本中,qRT-PCR检出甲型流感病毒21例,阳性率为52.5％;DIEFA检出甲型流感病毒22例,检出率为55.0％;GICA检出甲型流感病毒12例,检出率为30.0％.与qRT-PCR比较,DIEFA的灵敏度、特异性和一致性分别是85.7％、78.9％和82.5％,而GICA的灵敏度、特异性和一致性分别是42.8％、84.2％和62.5％.DIEFA对甲型流感病毒检出的灵敏度明显优于GICA(P＜0.05).结论 DIEFA方法检测甲型流感病毒具有灵敏度高及特异性好的优势,适用于流感流行季节对甲型流感病毒的快速诊断和筛查.     

甲型流感病毒NP及相关宿主因子研究进展

甲型流感病毒(influenza A virus, IAV)严重威胁人类健康。病毒核心是核糖核蛋白复合物(viral ribonucleoprotein complexes, vRNPs),是病毒基因组转录和复制的最小功能单位。核蛋白(nucleoprotein, NP)是vRNP的重要组成部分之一,在流感病毒整个生命周期起着关键作用。机体存在多种宿主因子通过NP影响病毒增殖。本文就通过NP影响vRNP核输入、病毒基因组转录和复制、vRNP核输出以及vRNP组装这几个方面的宿主因子进行综述,以期为治疗IAV新靶点的发现及新药物的研发提供参考。     

金标渗滤法快速检测血吸虫循环抗原的现场应用研究

将抗重组蛋白ＳＶＬＢＰ－ＩｇＧ用于ＤＩＧＦＡ检测２２２１例血清中血吸虫循环抗原,其中急性血吸虫病人血清７０份,慢性血吸虫病人血清３０７份,循环抗原检出率分别为１００％和６８．４％。非流行区健康人血清２００份,未见阳性反应。除肺吸虫外,与其它寄生虫或非寄生虫感染病人血清无明显交叉反应。用ＤＩＧＦＡ单盲法检测２批血清,结果显示对急性血吸虫病人的敏感性均为１００％;对慢性血吸虫病人的敏感性分别为６９．４     

一种唾液快速检测HIV-1/2型抗体试剂的现场评价

目的对一种唾液快速检测艾滋病病毒Ⅰ/Ⅱ型(HIV-1/2)抗体试剂进行现场评价,考核其现场使用的敏感性和特异性,以及与血液HIV-1/2抗体检测试剂的一致性。方法采集247例已知HIV感染者、1 090例正常献血人员、60例患有其它疾病的患者(包括孕妇、肿瘤患者、一般疾病患者),以及109例HCV感染者和119例未知HIV感染状况的吸毒人员的唾液标本,现场使用Vanguard OMTTM唾液HIV-1/2抗体快速检测试剂盒(CalypteBiomedical生产)进行检测,同时平行采集上述人群的血液标本,使用Vironostika HIV Uni-FormⅡplus O(BioMerieux生产)进行对比测试。结果247例已知HIV感染者中,247例唾液标本HIV-1/2型抗体检测均为阳性。1 259例血液酶联免疫吸附试验(ELISA)检测HIV抗体阴性人群中,1 257例唾液检测为阴性,2例为阳性。119例吸毒人员中,28例血液标本HIV阳性中,唾液标本检出27例。91例HIV阴性中检出阴性90例。该唾液HIV抗体快速检测试剂,敏感性为99.64%,特异性为99.78%。与ELISA检测的一致性为99.75%。结论唾液HIV-1/2型抗体检测试剂与现行的血液ELISA检测结果相近。唾液标本的采集方便而且危险性小,推荐在采血较困难的人群、基层医疗机构、VCT门诊等,可考虑使用唾液HIV-1/2型抗体快速检测试剂进行HIV抗体初筛检测。     

免疫胶体金渗滤法快速诊断试剂盒对包虫病患者血清的检测

囊型包虫病又称囊型棘球蚴病 ,是人类感染细粒棘球绦虫的幼虫 (细粒棘球蚴 )所致的一种慢性寄生虫病 ,棘球蚴可以寄生在人体的任何部位。医学影像学是目前诊断本病的可靠方法。血清抗体的检测也有一定的诊断价值。我院从1999- 2 0 0 3年共有 2 4例经彩超诊断和手术证实的囊型包虫病患者 ,现将采用新疆兆丰生物技术工程公司生产的免疫胶体金渗滤法———快速诊断试剂盒检测这些患者血清的结果见表 1:经过统计分析在 2 4例经手术证实的包虫病人中 ,用本试剂进行血清学检测的敏感度为 5 0 %。其中男性患者血清检测的敏感度较高 ,为 6 2 .5 % ,…     

H5N1亚型禽流感病毒核蛋白NP的原核表达及抗体制备

目的制备H5N1亚型禽流感病毒NP重组蛋白和抗体,为研制含NP组分的人禽流感亚单位疫苗和建立评价方法提供检测抗原和抗体。方法以H5N1亚型禽流感病毒A/chicken/Hubei/489/2004(H5N1)NP基因cDNA克隆质粒pMD-NP为模板,PCR扩增获得NP基因抗原区片段,克隆到原核表达载体pET-28a,构建重组质粒pET-ΔNP,转化大肠杆菌BL21-CondonPlus(DE3)-RIL,IPTG诱导重组蛋白表达。采用Ni亲和层析方法纯化NP重组蛋白,免疫家兔,收集免疫血清,ELISA测定抗体效价;间接免疫荧光检测所制备抗体与真核细胞表达NP蛋白的反应性。结果与结论成功的制备了高效价兔抗NP抗体,抗体效价在105以上,该抗体能与原核和真核系统中表达的NP蛋白特异性反应,为研制以NP蛋白为抗原组分的人禽流感亚单位疫苗和建立评价方法奠定了基础。     

新的“芯片”可以快速检测禽流感病毒

据Medscape．com 11月8日报道，美国研究人员指出，一种新的“芯片”可以检测11种不同的流感病毒株，包括禽流感病毒。在15分钟之内可以诊断出流感患。该项研究仍在进行中，研究人员希望把它发展成能够现场检测流感病毒的技术。     

快速检测抗日本血吸虫抗体的金标免疫渗滤法的建立及应用

以血吸虫虫卵浸出液为抗原，金标记兔抗人ＩｇＧ为显示剂，建立了检测抗血吸虫抗体的金标免疫渗滤法（ＤＩＧＦＡ）。以该法检测血吸虫病患者血清８７份，阳性检出率为９８．９％，非流行区血清１０份，阴性符合率为１００％，１５例肺吸虫病患者血清，１例出现阳性反应，交叉反应率为６．７％；１０例华支睾吸虫病患者和２５例乙型肝炎患者，均无交叉反应。本法与ＥＬＩＳＡ比较，符合率达９６．７％，揭示ＤＩＧＦＡ的敏感性和特异     

禽流感病毒NP基因的酵母双杂交饵载体表达及自激活检测

目的构建H5N1亚型禽流感病毒NP基因的酵母双杂交诱饵载体,验证其在酵母中的表达并检测其自激活作用。方法以PCR法从pGEMT/H5NP扩增NP基因编码区序列,将其定向克隆到PGBKT7载体,经测序鉴定后,PEG/Li-Ac法转化酵母菌株AH109,用表型筛选法及颜色筛选法检测其自激活作用同时Westernblot验证诱饵蛋白的表达。结果获得NP编码区基因,并成功构建酵母双杂交系统中的诱饵载体pGBKT7-NP,对宿主细胞酵母菌株AH109无毒性和自激活作用,并能在酵母细胞中稳定表达。结论诱饵载体pGBKT7-NP可用于GAL4酵母双杂交系统钓取与禽流感病毒核蛋白相互作用的蛋白。     

Analytical detection of influenza A(H3N2)v and other A variant viruses from the USA by rapid influenza diagnostic tests

Please cite this paper as: Balish et al. (2012) Analytical detection of influenza A(H3N2)v and other A variant viruses from the USA by rapid influenza diagnostic tests. Influenza and Other Respiratory Viruses 7(4), 491–496. Background The performance of rapid influenza diagnostic tests (RIDTs) that detect influenza viral nucleoprotein (NP) antigen has been reported to be variable. Recent human infections with variant influenza A viruses that are circulating in pigs prompted the investigation of the analytical reactivity of RIDTs with these variant viruses. Objectives To determine analytical reactivity of seven FDA‐cleared RIDTs with influenza A variant viruses in comparison with the reactivity with recently circulating seasonal influenza A viruses. Methods Tenfold serial dilutions of cell culture–grown seasonal and variant influenza A viruses were prepared and tested in duplicate with seven RIDTs. Results All RIDTs evaluated in this study detected the seasonal influenza A(H3N2) virus, although detection limits varied among assays. All but one examined RIDT identified the influenza A(H1N1)pdm09 virus. However, only four of seven RIDTs detected all influenza A(H3N2)v, A(H1N2)v, and A(H1N1)v viruses. Reduced sensitivity of RIDTs to variant influenza viruses may be due to amino acid differences between the NP proteins of seasonal viruses and the NP proteins from viruses circulating in pigs. Conclusions Clinicians should be aware of the limitations of RIDTs to detect influenza A variant viruses. Specimens from patients with influenza‐like illness in whom H3N2v is suspected should be sent to public health laboratories for additional diagnostic testing.     

Detection of 2009 pandemic influenza A(H1N1) virus Infection in different age groups by using rapid influenza diagnostic tests

Please cite this paper as: Gao et al. (2011) Detection of 2009 pandemic influenza A(H1N1) virus Infection in different age groups by using rapid influenza diagnostic tests. Influenza and Other Respiratory Viruses 6(3), e30–e34. Background The performance of rapid influenza diagnostic tests (RIDTs) in detecting influenza A(H1N1) 2009 has varied widely. Evaluations of RIDTs among infected individuals across all age groups have not been described in depth. Objectives Determine RIDT clinical sensitivity in comparison with influenza detection using real‐time RT‐PCR among patients infected with influenza A(H1N1) 2009 across all age groups. Study design This study analyzed respiratory specimens received by the New Hampshire Public Health Laboratories (NHPHL) from September 1, 2009, through December 31, 2009. RIDT performance was evaluated among different age groups of patients determined to be infected with influenza A (H1N1) 2009, and the association between age and RIDT sensitivity was determined. Results Of 1373 specimens examined, 269 tested positive for influenza A(H1N1) 2009 by real‐time RT‐PCR (rRT‐PCR) and had RIDT results available. Overall clinical sensitivity and specificity of RIDTs were 53·9 and 98·5%, respectively. By age group, clinical sensitivity was 85·7% in patients <2 years old, 60·3% in patients between 2‐ and 39 years old, and 33·3% in patients aged 40 and older. Logistic regression analysis indicated that increasing age was negatively associated with RIDT performance. Conclusion Rapid influenza diagnostic test sensitivity decreased significantly with increasing age. Findings from this study may impact a clinician’s interpretation of RIDT test results and ultimately have implications in clinical decision‐making.     

Molecular genetic characteristics of influenza A virus clinically isolated during 2011‐2016 influenza seasons in Korea

Background

The influenza virus is reportedly associated with 3‐5 million cases of severe illness and 250 000‐500 000 deaths annually worldwide.

Objectives

We investigated the variation of influenza A virus in Korea and examined the association with death.

Methods

A total of 13 620 cases were enrolled in the Hospital‐based Influenza Morbidity & Mortality surveillance system in Korea during 2011‐2016. Among these cases, a total of 4725 were diagnosed with influenza using RT‐PCR (influenza A; n = 3696, influenza B; n = 928, co‐infection; n = 101). We used 254 viral sequences from the 3696 influenza A cases for phylogenetic analysis using the BioEdit and MEGA 6.06 programs.

Results

We found that the sequences of A/H3N2 in the 2011‐2012 season belong to subgroup 3C.1, whereas the sequences in the 2012‐2013 season pertain to subgroup 3C.2. The sequences in the 2013‐2014 and 2014‐2015 seasons involve subgroups 3C.3a and 3C.2a. The A/H1N1pdm09 subtype belongs to subgroup 6 and contains two clusters. In addition, sequence analysis confirmed the several substitutions of internal genes and gene substitutions associated with drug resistance (I222V in NA and S31N in M2) in the fatal cases. While statistical analysis found no significant associations between genetic differences in the viruses and mortality, mortality was associated with certain host factors, such as chronic lung disease.

Conclusions

In conclusion, influenza A virus clade changes occurred in Korea during the 2011‐2016 seasons. These data, along with antigenic analysis, can aid in selecting effective vaccine strains. We confirmed that fatality in influenza A cases was related to underlying patient diseases, such as chronic lung disease, and further studies are needed to confirm associations between mortality and viral genetic substitutions.     

SERS-based immunomagnetic bead for rapid detection of H5N1 influenza virus

The surface-enhanced Raman scattering (SERS) has recently drawn attention in the detection of respiratory viruses, but there have been few reports of the direct detection of viruses. In this study, a sandwich immunomagnetic bead SERS was established for the rapid diagnosis of the H5N1 influenza virus. The detection limit was estimated to be 5.0 × 10⁻⁶ TCID₅₀/ml. The method showed excellent specificity with no cross-reaction with H1N1, H5N6 or H9N2. The H5N1 influenza virus detection accuracy of the SERS method was 100% in chicken embryos. The results hold great promise for the utilization of SERS as an innovative approach in the diagnosis of influenza virus.     

人禽流感死亡病例不同组织器官中H5N1病毒的检测分析

目的了解H5N1病毒在人禽流感死亡病例组织器官中的分布和含量。方法在BSL-3实验室，测定H5N1禽流感病毒分离株的TCID50，并采用荧光定量PCR制作标准曲线；将人禽流感死亡病例的尸解标本，包括心、肝、脾、肺、肾、脑等组织标本研磨处理后，进行荧光定量PCR检测，并根据标准曲线推算H5N1禽流感病毒的病毒载量。结果心、肝、肾、脑组织标本中H5N1禽流感病毒检测为阴性，在肺、脾组织标本中检测到H5N1病毒，病毒载量分别为10^6.3TCID50／ml和10^4.55TCID50／ml。结论除呼吸系统外，在脾组织中也存在H5N1禽流感病毒。     

多重反转录聚合酶链反应快速检测鉴别H9亚型禽流感病毒方法的建立

目的参考AIV M基因和HA基因序列,设计了两对引物。其中XZ145-2和XZ146为通用引物,可以检测所有亚型AIV,跨幅244 bp;XZ H9-1和XZ H9-2为H9亚型特异性引物,跨幅488 bp。方法利用这两对引物,通过对多重RT-PCR扩增条件的优化,成功建立了快速检测鉴别H9亚型AIV的多重RT-PCR技术。结果与结论特异性和敏感性试验结果表明,该技术对H9亚型AIV同时扩增出两条大小分别为244 bp和488 bp的cDNA片段,对其他亚型AIV只扩增出244bp的cDNA片段,对其他常见禽病病原无特异性扩增,结果为阴性;该多重RT-PCR的通用引物对AIV RNA的最低检出量为1pg,对H9亚型RNA的最低检出量为10 pg。     

Detection of oseltamivir‐resistant zoonotic and animal influenza A viruses using the rapid influenza antiviral resistance test

Mutations in the influenza virus neuraminidase (NA) that cause reduced susceptibility to the NA inhibitor (NAI) oseltamivir may occur naturally or following antiviral treatment. Currently, detection uses either a traditional NA inhibition assay or gene sequencing to identify known markers associated with reduced inhibition by oseltamivir. Both methods are laborious and require trained personnel. The influenza antiviral resistance test (iART), a prototype system developed by Becton, Dickinson and Company for research use only, offers a rapid and simple method to identify such viruses. This study investigated application of iART to influenza A viruses isolated from non‐human hosts with a variety of NA subtypes (N1‐N9).