Objective:To establish a quick on-site emergency detection method for severe fever with thrombocytopenia syndrome virus (SFTSV), dengue virus (DENV), and hantaan virus (HTNV).Methods:This research was based on the traditional TaqMan fluorescent probe technology, using the domestic rapid one-step quantitative RT-PCR kit, combined with the Magnetic induction cycler (Mic) qPCR instrument. The detection limit, specificity and repeatability of this method were evaluated by simulated samples, other virus infected samples and normal human blood samples.Results:Compared with the traditional RT-PCR assay, the required time of this method was greatly shortened, and the detection can be completed within 35 minutes. The limit of quantitation for SFTSV, DENV and HTNV are less than 100copies/PCR. No nonspecific amplification was found in the simulated negative samples and other virus infected samples. All the simulated positive sample for verification could be detected, and coefficient of variation Ct value of each group was less than 4%. Conclusions:The rapid fluorescence quantitative RT-PCR assays have certain application prospects for on-site emergency detection, and provide important technical supports and new directions for the prevention and control of common hemorrhagic fever viruses.

Objective:To establish a simple, rapid and low-cost 2019 novel coronavirus (2019-nCoV) neutralizing antibody detection method.Methods:The 2019-nCoV RBD specific immunoglobulin G (RBD-IgG) detection method was established based on the principle of quantum dot immunochromatography(QDs), and the detection was evaluated by using of sera from coronavirus disease 2019 (COVID-19) convalescent patients ( N = 97), vaccinated donors ( N = 82) and healthy donors ( N = 299). The suitability of fingertip blood was evaluated by matching blood samples with peripheral blood ( N=54). Results:The 2019-nCoV RBD-IgG detection method based on QDs was successfully established. The detection result of QDSs had strong correlation ( Spearman r > 0.73, P < 0.000 1) and good consistency ( Kappa=0.93, P < 0.01) with the result of micro-neutralization test(MNT). The sensitivity and specificity were 92% and 99%, respectively. There was high correlation ( Spearman r =0.932 6, P < 0.000 1) and no significant difference ( P=0.102 6) between result of fingertip blood and peripheral blood. Fingertip blood can be used as a surrogate sample for testing. Conclusions:The 2019-nCoV neutralizing antibody detection method established in this study can provide an immediate, efficient and low-cost method selection for the assessment of herd immunity status, and provide technical support for the herd immunity monitoring of 2019-nCoV vaccinated population and the prevention and control of the epidemic.

Objective:To explore an early, rapid and accurate detection method of severe acute respiratory syndrome coronavirus 2 (2019-nCoV) antigen and improve the detection rate of patients with 2019-nCoV.Methods:We detected the characteristics of monoclonal antibodies (mAbs) against 2019-nCoV generated previously and established a double antibody sandwich ELISA against S protein of the virus.Results:All 12 mAbs against 2019-nCoV screened in our previous study were able to recognize purified virion and S protein. They were good candidates for detecting antibody to 2019-nCoV antigen. A double antibody sandwich ELISA established in this study could rapidly and effectively detect 2019-nCoV S protein with high sensitivity. The detection limit of 2019-nCoV S protein was lower than 1 ng/ml.Conclusions:This method could specifically detect 2019-nCoV antigen, and provided a meaningful reference for early, sensitive and specific diagnosis of COVID-19 infection.

Flexal病毒(Flexal virus,FLEV)和Whitewater Arroyo病毒(Whitewater Arroyo virus,WWAV)属沙粒病毒,经鼠传播,可引起人类严重传染病,主要分布于美洲,有因野外工程而感染或国际物流和人员往来而输入的风险.本研究通过检索、分析FLEV和WWAV基因组序列,参照同科属病毒遗传进化特征,确定病毒基因组保守区,筛选分子检测靶基因,设计PCR扩增引物和检测探针,建立两种沙粒病毒的实时荧光RT-PCR检测方法,并进行灵敏性、特异性和重复性初步评价.结果 表明,所建立的FLEV和WWAV检测方法与其他病毒无交叉反应,扩增效率大于95％,检出限分别为100copies/μL和20copies/μL,变异系数在2％以内.上述结果显示,本研究所建立的FLEV和WWAV检测方法的特异性强,灵敏性与重复性均处于常规病毒性传染病分子诊断可接受范围,可应用于疑似病例的快速检测和相关病原的筛查和流行病学调查.

致病性汉坦病毒的宿主主要为啮齿类动物,其病毒感染状况是人间疫情发生的关键影响因素,可通过检测宿主动物标本中病毒基因组RNA、蛋白抗原及特异性抗体而进行监测.本研究利用367份鼠肺及鼠血标本,对双抗原夹心ELISA (ELISA)、实时荧光RT-PCR(RT-PCR)和免疫荧光(IFA)等三种分别检测抗体、核酸和抗原的方法进行比较评估.ELISA法检出抗体阳性鼠血标本46份,阳性率为12.53％;RT-PCR法检出病毒RNA阳性鼠肺标本28份,阳性率为7.63％;IFA检出抗原阳性鼠肺标本24份,阳性率为6.54％.宿主动物组织标本中检出汉坦病毒RNA和(或)结构蛋白抗原的标本,对应的血液标本中可检出病毒特异性抗体,100％ (24/24)IFA检测阳性标本和89.3％ (25/28) RT-PCR检测阳性标本对应血标本ELISA抗体检测阳性,反之亦然,检出抗体的标本基本包含了可检出抗原和RNA的标本.RT-PCR与IFA检测结果差异无显著性(x2a=0.64,P＞0.05),一致性检验Kappa系数为0.71,一致性高(Z=13.66,P＜0.05),首先对血标本开展基于ELISA的特异性抗体检测,可显著缩小RT-PCR或IFA法检测病毒RNA或抗原的范围(x2b=12.04,x2c=20.05,P＜0.05).本研究为宿主动物汉坦病毒感染实验室监测方案优化提供了有益的依据.

目前国内外针对寨卡病毒(Zika virus,ZIKV)的研究多集中于E蛋白与NS1蛋白,而对于其他非结构蛋白的研究则相对较少.为了进一步了解ZIKV感染免疫机制,为ZIKV免疫学检测和流行病学研究提供新的工具方法,本研究建立了基于NS2B蛋白多肽抗原荧光素酶免疫吸附试验(Luciferase immunosorbent assay,LISA)的新方法,应用于检测抗ZIKV IgG抗体(anti-ZIKV IgG).通过构建NS2B蛋白特异性多肽抗原与新型荧光素酶融合表达的载体,转染真核细胞后获得含有目标抗原与荧光素酶融合蛋白的细胞裂解物,建立anti-ZIKV IgG检测的LISA法.采用正常人血清、ZIKV感染的阳性血清、乙脑病毒(Japanese encephalitis virus,JEV)感染阳性血清,登革病毒(Dengue virus,DENV)感染阳性血清,评价该方法的特异性、灵敏度与重复性及交叉反应,并与基于NS1纯化抗原的商业化ELISA试剂盒(NS1-ELISA)进行平行比较.成功建立的NS2B-LISA方法,检测灵敏度和特异度分别为92％和96.2％,通过阳性样本倍比稀释评价两种检测方法灵敏度,NS2B-LISA的灵敏度比NS1-ELISA高16倍.重复性实验显示NS2B-LISA法检测anti-ZIKV IgG板间变异系数为(3.1±0.7)％,板内变异系数为(2.6±0.9)％.本研究基于NS2B蛋白多肽抗原成功建立了anti-ZIKV IgG检测的LISA新方法,该方法简便、特异、灵敏、重复性好,易于高通量自动化,可用于anti-ZIKV IgG感染的免疫学诊断与流行病学研究.

Objective To establish a method for the simultaneous identification of Zika, Chikungunya and Mayaro viruses. Methods The complete genome sequences of Zika, Chikungunya and Mayaro virus were retrieved from Global Shared Database for comparative analysis, estimate its conservative region and determine the target gene location, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity, sensitivity and repeatability of the assay were assessed by viral nucleic acid of Zika virus, Chikungunya virus a, in vitro transcriptional RNA of Mayaro virus, normal human serum and related virus simulation sample. Results The result showed that the established method could detect Zika virus, Chikungunya virus, as well as simulated Mayaro virus samples, the limit of detection (LOD) of Zika and Chikungunya virus was 16.22 Copy/PCR and 12.02 Copy/PCR, respectively, the LOD for simulated Mayaro virus RNA was 2.82 Copy/PCR, no significant difference was detected between the triplex and monoplex assays. No cross reaction was found in the detection of dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome (SFTS) virus, yellow fever virus and influenza virus, and 100 healthy adults blood samples, the specificity of the method was 100%. The repeatability result showed that the standard deviation of all three detections were blow 0.5 and the coefficient of variation was less than 2% by selecting viral nucleic acids or transcribed RNA with high, medium and low concentration gradients. Conclusions A triplex real-time RT-PCR assay for detection of Zika, Chikungunya and Mayaro virus has been established with an acceptable specificity, sensitivity and repeatability. Key words: Zika virus; Chikungunya virus; Mayaro virus; Triplex Real-time RT-PCR

目的 了解拉沙热在塞拉利昂西区流行和分布情况.方法 用随机抽样的方式采集并检测了820份塞拉利昂西区人群血清中的拉沙热IgG抗体,并分析其分布特征.结果 总体拉沙热IgG抗体阳性率为28.78％,95％可信区间(Confidence Interval,CI)为25.68％～31.88％.拉沙热阳性率西区郊区43.48％(80/184),高于西区城市24.82％(136/548)(x2=23.06,P＜0.05),差异有统计学意义;不同性别和不同年龄组之间拉沙热阳性率差异无统计学意义.结论 塞拉利昂西区拉沙热总体阳性率较高,且郊区和城市差异明显,应加强环境卫生的改善,加大防鼠灭鼠工作.

Objective To clarify the potential pathogen for fever of unknown origin (FUO) in serum samples for which pathogenic agents were hardly identified with conventional exainatins.Methods Random capturing the nucleic acid of pathogen was performed by utilizing the property of sequence nondependence of next generation sequencing (NGS),followed by enrichment of nucleic acid with multiple displacement amplification (MDA).After sequencing,metagenomic analysis was applied to the raw data and the phylogenetic tree was built to identify the potential pathogen.Results The result did not indicate common pathogens for FUO but showed the existence of Torque teno Viurs (TTV).Assembly was carried out to all sequencing reads.The coverage of consensus sequence on reference was calculated.Phylogenetic result indicated that all confidence sequences belonged to 3 genera (α TTV,β TTV and γ TTV).Conclusions The characteristics of genome,phylogenesis of TTV and TTV as signal at immunology level were analyzed and clarified.The possible explanation for detection of TTV in 3 genera may be that TTV itself or noninfectious factors caused the immunosuppression,which finally result e in rise of TTV detection.

To study the specific IgA antibody levels in serum, urine and saliva samples from Zika virus (ZIKV) infected patients, and to further understand the mechanism of the immune response to ZIKV infections, the recombinant ZIKV NS1 protein was expressed and purified. The concentration and purity of the protein were identified and its antigenicity was preliminarily evaluated. The indirect enzyme linked immunosorbent assay (Indirect-ELISA) was used to detect the specific IgA antibody in serum, urine and saliva samples from ZIKV infected patients. The specificity was evaluated using the serum, urine and saliva samples from healthy volunteers. The cutoff value was determined with the mean optical density value for the negative control plus three times the standard deviations. The specificity was 100％. Thirty-three serum samples, four urine samples and three saliva samples collected from ZIKV nucleic acid confirmed cases were detected. Three serum samples with high IgA antibody level were selected and serially diluted twofold to evaluate the specific IgA antibody titer. It is shown that the IgA antibody in 3, 200 times diluted serum could be detected effectively. The repeatability showed that the coefficient of variation (CV) between the plates was (3±0. 8) ％, and the CV within plate was (2. 7±1. 0) ％. The detection rates of IgA antibodies in sera, urine and saliva samples were 75. 8％ (25/33), 100％ (4/4) and 33. 3％ (1/3), respectively. It is indicated that the high IgA level in three kinds of body fluids could be a detection marker for in vitro diagnosis. Meanwhile, the presence of specific IgA antibody in urine and saliva samples indicates potential mucosal immunity, which is helpful for understanding the viral spread and transmission in vivo and in vitro. It also demonstrated that the ZIKV NS1 protein could be used for the research and development of immunological diagnostic reagents for Zika virus.

An indirect enzyme-linked immunoassay (ELISA)-based serological method for detection of IgG antibodies to the Zika virus was established.Four types of Zika-virus antigen (ectodomain of E protein;NS1 protein;rEⅢ protein;C protein) were expressed,purified and coated on ELISA plates.Indirect ELISA methods were used for the sera of Zika-infected patients and those of healthy subjects to determine the sensitivity and specificity of each type of coated antigen.The concentration and purity of four Zika an tigens met the criteria for detection.The results of indirect ELISA showed that the sensitivity and specificity of detection for anti-E (ectodomain) and anti-NS1 IgG antibodies were high.However,the sensitivity of detection for anti-rEⅢ and anti-C antibodies was very low,so this method was not suitable for detection of virus antibodies.By evaluating the sensitivity and specificity of detection for specific IgG antibodies of four Zika antigens,we have laid the foundation for the development of diagnostic agents.

Objective To analysis the epitope of Ebola virus (EBOV) nucleoprotein (NP) and understand its immunological properties.Methods Firstly four mouse mAbs recognizing the different epitopes of Ebola virus NP were classified by competition ELISA.The full-length NP and the C-terminal were truncated and expressed in E.coli.according to epitope prediction by software and some published papers.Epitope mapping of mAbs to a panel of the truncated rNP were identified in WB analysis.Results After epitope prediction,the full-length NP were divided into rNP1-418,rNP419-639 and rNP640-739.WB analysis indicated that mAbs all recognized the linear epitopes,which were limited in the C-terminal 100aa (rNP640-739).The truncated N proteins with N-terminal of rNP640-739 series 20aa deletions reacted with all mAbs.The epitopes recognized by anti-NP mAbs were defined near the C-terminal 20aa of EBOV NP.Conclusion Analysis of antigenic epitopes of EBOV NP by specific mAbs as a probe lay the foundation for the study of immunology diagnostic reagents.

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

1 简介 生化分析是生物基础研究及医学临床诊断的重要手段,其中有30多年历史的流式细胞分析技术已广泛应用于生物医学基础研究及实际应用的各个领域.尽管在流式分析技术的大多数实际应用中都以细胞作为载体或研究目标,但诸如微球之类的小固态颗粒同样也可以应用于流式分析技术.