8种脑炎相关虫媒病毒GeXP检测方法的初步建立

利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(mRT-PCR)方法,同时检测与病毒性脑炎相关的乙型脑炎病毒(Japanese encephalitis virus,JEV)等8种虫媒病毒。优化多重反应体系及反应条件,分别以病毒分离培养物和阳性标本来验证多重反应体系的特异性,以克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出各病毒对应的特异片段,并可在102拷贝/μL水平同时并特异地检测出8种(共13个特异片段)脑炎相关病毒RNA。该方法具有高通量、灵敏度高、特异性强且快速等优点,对病毒性脑炎的分子诊断及流行病学调查具有重要意义。     

同时鉴别6种鸡病毒性呼吸道病GeXP检测方法的建立

为了建立一种能够同时鉴别H5、H7、H9亚型禽流感、新城疫、传染性支气管炎和传染性喉气管炎6种鸡病毒性呼吸道传染病病原体的GeXP检测方法,本研究根据这些病原体各自的基因保守序列,设计合成了7对特异性引物,优化反应条件,用单一病毒、混合病毒样品来验证所建立的GeXP方法的特异性和准确性,以不同拷贝数的克隆质粒来检测GeXP方法的灵敏度。检测34份临床阳性样品,进一步验证该法的可靠性。结果显示,在7对引物同时存在的GeXP反应体系中,可特异性地扩增出相关6种病毒目的片段,并可在102拷贝/μL水平同时特异地检测出鸡6种病毒性呼吸道传染病,GeXP方法检测34份临床阳性样品结果与病毒分离一致。本研究建立的GeXP方法不仅可高通量检测6种病原体,而且具有特异性强和灵敏度高的特点,适用于临床样品混合感染的快速鉴别诊断,对这6种发病特征及临床症状相似的鸡病毒性呼吸道病的有效防控有很高的实际应用价值。     

GeXP多重基因表达遗传分析系统在手足口病病原分型检测中的应用

利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(RT-PCR)方法,同时检测引起手足口病的9种常见的人肠道病毒—人肠道病毒71型(HEV71)、柯萨奇病毒A组(CVA)16、4、5、9、10型和柯萨奇病毒B组(CVB)1、3、5型。优化多重反应体系中针对5’UTR区的肠道病毒通用引物和11对针对9种血清型人肠道病毒VP1区的特异性引物的浓度比例,分别以病毒细胞培养物和阳性粪便标本来验证多重反应体系的特异性,以TCID50定量的细胞培养物和克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出人肠道病毒共有的保守片段的和型特异性片段,HEV71和CVA16细胞培养物的检测下限为100.5TCID50/μL,并可在103copies/μL水平同时、特异地检测出9种病毒RNA。该方法灵敏度高、特异性强,可快速对大量临床样本进行高通量检测,用于手足口病的分子流行病学调查。     

五种主要上呼吸道病毒多重RT-PCR 检测方法的建立

目的:建立甲型、乙型流感病毒、呼吸道合胞病毒A型、B型(RSV-A、RSV-B)和腺病毒(ADV)五种主要上呼吸道病毒的多重RT-PCR检测方法。方法:利用Primer premier5.0分别针对甲型流感病毒的M基因、乙型流感病毒的PB1基因、RSV-A和RSV-B的F基因及ADV的hexon基因设计五对特异性引物,对Mg2+、dNTP、引物浓度及退火温度等进行优化,建立同时检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV的多重RT-PCR方法,并验证该检测方法的灵敏性。结果:所建立的五种病毒的多重RT-PCR方法可以同时或者分别扩增甲型、乙型流感病毒、RSV-A、RSV-B及ADV的141bp、635bp、525bp、377b和283bp基因片段,敏感度分别达到770PFU/ml、800PFU/ml、680PFU/ml、970PFU/ml和850PFU/ml,且五种病毒间无交叉反应。结论:所建立的多重RT-PCR方法可以迅速准确地检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV,为五种病毒的检测提供了一种方便易行的方法。     

常见呼吸道病毒多重PCR检测法的质量控制分析

急性呼吸道感染广泛分布于人群中,主要由呼吸道病毒引起,在老年人和婴幼儿中具有较高的发病率和死亡率。快速准确检测出病原体对临床早期诊断和指导治疗有重要意义。本文通过分析16种常见呼吸道病毒多重PCR检测法的质量控制关键环节和质量控制措施,对有效加强实验室质量管理,从而为提高实验室核酸检测质量水平提供了基本的参考数据。     

七重呼吸道病毒液相芯片核酸检测技术的初步建立

由呼吸道病毒引起的疾病严重威胁着人类的生命健康,为了建立一种快速高通量的呼吸道病毒核酸检测方法,本研究将多重PCR技术同液相芯片技术结合起来,针对呼吸道合胞病毒A型和B型、乙型流感病毒Victoria系和Yamagata系、甲型流感病毒H1型和H3型以及新冠病毒等常见的七种呼吸道病毒,初步建立了七重呼吸道病毒液相芯片核酸检测技术,评价了方法的特异性、敏感性和重复性,并使用来自安徽省疾控的25份临床急性期样本核酸对方法进行验证。结果显示,建立起的基于液相芯片多重核酸检测方法可特异性识别七种目标呼吸道病毒的靶基因序列,与包括副流感病毒在内的9种非目标呼吸道病毒无交叉反应。对七种病毒核酸进行十倍稀释液相检测,其中H3、BV、RSVB可以检出10²拷贝/μL,BY、RSVA和SARS-CoV-2可以检出10³拷贝/μL,对H1的检测限为10⁴拷贝/μL。25份样本核酸检测结果与实际相符。结果表明,本研究建立的七重呼吸道病毒液相芯片核酸检测技术具有特异性强、敏感性高、稳定性好等特点,可用于临床样本的快速检测,为呼吸道类传染病的液相...     

建立新型的常见腹泻相关病毒的多重检测方法

利用GenomeLab(tm)GeXP遗传分析系统建立一种同时检测A组轮状病毒、诺如病毒GI、GII型、札如病毒、肠道腺病毒、星状病毒、人博卡病毒II型7种常见腹泻相关病毒的方法。对反应条件进行优化后,非同日三次重复实验表明至少在104拷贝/μL水平可同时特异地检测出7种病毒,对Enterovirus71、Human Parechovirus、Picobir-navirusII阳性标本无交叉反应。本研究初步建立了一种高通量、快速的常见腹泻相关病毒的检测方法,为腹泻病原的分子诊断提供了新的方法。     

新型多重PCR方法及其在呼吸道病毒诊断上的应用

急性呼吸道感染广泛分布于人群中,主要由呼吸道病毒引起,在老年人和婴幼儿中具有较高的发病率和死亡率,给社会带来严重的经济负担。快速、准确检测出病原体对临床早期诊断和指导治疗有重要意义。多重聚合酶链式反应(PCR)技术具有PCR的高灵敏度和快速检测的特点,还能实现多病原的高通量检测。基于多重PCR技术开发的商业化试剂盒能同时检测12种以上的呼吸道病毒,能达到与实时荧光定量PCR(real-time PCR)相当的灵敏度和特异性,为呼吸道病毒的检测与分型提供了新的选择。本文综合了近年来的发展成果,对新型多重PCR方法的原理及其在呼吸道病毒诊断上的应用进行了综述。     

采用基因微阵列技术对常见呼吸道病毒进行检测的初步研究

建立一种高通量的基因微阵列检测技术,对常见呼吸道病毒感染进行监控.根据公开发表的8个病毒科38种常见呼吸道病毒的序列,计算其保守区域,设计病毒的特异性检测探针,制备呼吸道病毒检测基因微阵列.利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与基因微阵列上的探针杂交,清洗、扫描后进行结果分析.采用流感病毒、麻疹病毒、腮腺炎病毒和风疹病毒作为报告病毒,并对80例上呼吸道感染患者的咽拭子标本进行验证测试.初步结果表明,该呼吸道病毒微阵列基因芯片检测是可行的,在利用基因微阵列技术对病毒监控方面进行了有益的尝试,得到了有经验的信息.     

Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay

Background

Immunosuppressive viruses are frequently found as co-infections in the chicken industry, potentially causing serious economic losses. Because traditional molecular biology methods have limited detection ability, a rapid, high-throughput method for the differential diagnosis of these viruses is needed. The objective of this study is to develop a GenomeLab Gene Expression Profiler Analyser-based multiplex PCR method (GeXP-multiplex PCR) for simultaneous detection of eight immunosuppressive chicken viruses.

Results

Using chimeric primers, eight such viruses, including Marek's disease virus (MDV), three subgroups of avian leucosis virus (ALV-A/B/J), reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), chicken infectious anaemia virus (CIAV) and avian reovirus (ARV), were amplified and identified by their respective amplicon sizes. The specificity and sensitivity of the optimised GeXP-multiplex PCR assay were evaluated, and the data demonstrated that this technique could selectively amplify these eight viruses at a sensitivity of 100 copies/20 μl when all eight viruses were present. Among 300 examined clinical specimens, 190 were found to be positive for immunosuppressive viruses according to this novel assay.

Conclusion

The GeXP-multiplex PCR assay is a high-throughput, sensitive and specific method for the detection of eight immunosuppressive viruses and can be used for differential diagnosis and molecular epidemiological surveys.

     

Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco

Molecular Biology Reports - Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. In this...     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Background

Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples.

Results

Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples.

Conclusions

These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.