LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法的建立

目的 人副流感病毒1,2,3型是呼吸道感染的主要病原.本研究建立了特异、快速、灵敏的多重荧光定量RT-PCR方法用于人副流感1,2,3型病毒临床标本检测.方法 针对人副流感1,2,3型病毒设计特异性引物探针,优化荧光RT-PCR反应条件.应用体外转录方法分别制备人副流感1,2,3型病毒的标准品.验证荧光定量RT-PCR方法的特异性,敏感性和稳定性.结果 该方法对人副流感1,2,3型病毒核酸检测有高度特异性,检测的灵敏度HPIV1为10个拷贝,HPIV2为100个拷贝,HPIV3为100个拷贝.可从临床患者鼻咽吸出物标本中直接检出.结论 本研究建立的LNA探针同时检测人副流感病毒1,2,3型多重荧光定量RT-PCR方法具有较高的特异性和敏感性.适用于临床早期诊断和实验室病原谱筛查.     

马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立

目的 建立一种快速、敏感、特异的双重实时荧光定量PCR方法,可同时检测马尔堡病毒和埃博拉病毒.方法 通过序列比对挑选出两种病毒基因组中高度保守的序列,分别设计引物及Taqman探针,两条探针分别标记FAM和Texas Red荧光报告基因,建立双重实时荧光定量PCR反应体系.结果 双重荧光定量PCR方法检测两种病毒阳性标准品的灵敏度分别为30.5拷贝/μl和28.6拷贝/μl,通过检测日本脑炎病毒、黄热病毒、登革热病毒无交叉反应,有较好的灵敏度和特异性.结论 建立了马尔堡、埃博拉病毒双重荧光定量PCR检测方法,实现了两种病毒同时实时定量检测,在传染病防控领域有较好的应用前景.     

登革热病毒RNA实时荧光定量RT-PCR检测方法的建立

目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒（DV）及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。     

荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

目的：建立一种高效、简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测.方法：利用Bac-to-Bac载体系统在E.coli菌株DH10 Bac中构建重组杆状病毒穿梭质粒（Bacmid）和在昆虫细胞中构建含人IL-18基因的重组杆状病毒,纯化的重组Bacmid作为PCR检测的标准模板,由昆虫细胞中收获的病毒母液用于空斑测定和病毒DNA提取.以10倍梯度稀释的重组Bacmid作为标准模板,进行荧光定量PCR扩增IL18基因片段并绘制标准曲线,然后以提取的重组杆状病毒DNA作为模板,采用同样体系进行实时PCR反应检测.同时,以琼脂糖空斑法测定病毒母液的滴度.结果：成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法.运用标准模板进行的PCR反应显示该方法的线形范围为101～108拷贝,病毒母液的DNA拷贝浓度（vg/ml）值约为空斑检测的滴度pfu/ml值的10倍.结论：荧光定量PCR方法可灵敏快速地鉴定重组杆状病毒,并在较大的线性范围内检测重组杆状病毒滴度,较之空斑法更准确地反映了重组杆状病毒的实际数量.     

荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

目的：建立一种高效﹑简便的荧光实时定量PCR方法，用于重组杆状病毒鉴定及病毒滴度的检测。方法：利用Bac-to-Bac载体系统在昆虫细胞中构建含人IL-18基因的重组杆状病毒，收获的病毒母液以10倍梯度系列稀释后，提取病毒基因组DNA。以10倍梯度稀释的重组杆状病毒穿梭质粒（bacmid）作为标准模板，进行荧光定量PCR反应扩增IL-18基因片段并绘制标准曲线，然后以上述的重组杆状病毒基因组DNA作为模板，采用同样体系进行实时PCR反应检测，同时用琼脂糖空斑法测定病毒母液的滴度。结果：成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法。运用标准模板进行的PCR反应显示该方法的线形范围为101-108拷贝，病毒母液的DNA拷贝浓度（vg/mL）值约为空斑检测的滴度 pfu/mL值的10倍。结论：荧光定量PCR方法可灵敏快速地鉴定重组杆状病毒，并在较大的线形范围内检测重组杆状病毒滴度，较之空斑法更准确地反映了重组杆状病毒的实际数量。     

扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究

目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95％～105％,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10～100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.     

建立DNA芯片技术检测丙型肝炎病毒基因型及其初步应用

目的 建立一种简便、快速的以DNA芯片技术为基础的丙型肝炎病毒基因分型检测方法。方法 根据丙型肝炎病毒HCV的基因序列信息设计分型探针，将采用荧光标记的寡核苷酸引物所扩增的丙型肝炎病毒基因产物与结合在芯片上的特异性探针进行快速杂交，通过扫描荧光强度值定性和定量判定结果。结果 应用本法对65例患者血清中的丙型肝炎病毒核酸进行基因分型，对部分标本检测结果与测序法进行对比，吻合率达100％，说明检测结果有很高的特异性。结论 我们所用DNA芯片技术检测HC VRNA及其基因分型，简便、快速、特异性好、灵敏度高、结果判定指标客观，可望在临床推广使用。     

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.     

登革1型和2型病毒混合感染样本的一步法实时荧光定量PCR检测方法的建立

本文旨在建立检测登革1型和2型病毒的一步法实时荧光定量PCR方法,为登革热的诊断和预测提供技术支撑。依据登革1型和2型病毒的保守区序列设计引物和探针,建立了同时检测两个血清型登革病毒的一步法实时荧光定量PCR方法。特异性实验结果显示,该方法可检测出登1型和2型病毒,与登革3型、4型病毒、黄热病毒、流感H3N2病毒均无交叉反应,具有较好的特异性。对登革1型和登革2型的灵敏度均为102copies/μL。应用该方法,可检测出登革1型和2型病毒混合感染C6/36后不同时间点病毒复制动态。在两个血清型登革病毒混合感染C6/36细胞72~120 h登革2型病毒的拷贝数与单独感染时登革2型病毒的拷贝数相比显著下降。上述研究证实建立的一步法实时荧光定量PCR方法具有特异性好灵敏度高、重复性好等特点,能够用于登革型和2型病毒混合感染样本的检测。     

阿瓦朗病毒和休斯病毒实时荧光RT-PCR检测方法研究

目的建立可以检测阿瓦朗病毒(Avalon virus,AVAV)和休斯病毒(Hughes virus,HUGV)两种内罗病毒的实时荧光定量RT-PCR检测方法,并进行初步的评价。方法收集、整理、比对、分析在公共数据库发布的两种病毒基因组核苷酸序列,确定检测靶标,设计特异性引物、探针,优化检测程序,建立实时荧光定量RT-PCR检测方法。利用体外转录技术制备的模拟样本、其他病毒感染标本、病毒株和正常人血标本比较评价所建方法的检测限、特异性、重复性特征。结果所建实时荧光定量RT-PCR检测方法可有效扩增检测AVAV和HUGA靶标RNA,检测限分别约为20拷贝/μl和70拷贝/μl,检测科萨努尔森林病毒、乙型流感病毒BV和BY型、甲型流感病毒H3N2、黄热病毒、乙型脑炎病毒、克里米亚-刚果出血热病毒、发热伴血小板减少综合征、内罗毕羊病毒和塔西那病毒样本无非特异性扩增,两种内罗病毒相互间无交叉反应,重复性比较分析显示变异系数小于2%。结论本研究建立的检测AVAV和HUGV的实时荧光定量RT-PCR方法,可用于临床样本检测和媒介生物、宿主动物标本筛查,便于病原的快速识别和疾病诊断。     

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.     

Anchored Pan Dengue RT‐PCR and Fast Sanger sequencing for detection of Dengue RNA in human serum

A large number of human infections are caused by different dengue virus strains, mainly in the tropical and subtropical parts of the world, but also outside the endemic regions. RT‐PCR methods are used widely for detection of dengue virus RNA in acute‐phase serum samples; however, new sequence variation can inhibit these methods. An assay was developed integrating an anchored Pan Dengue RT‐PCR with a new Fast Sanger sequencing protocol. For broad detection and identification of dengue virus RNA, including new strains of all serotypes, the conserved 3′ genome end was targeted for highly specific cDNA synthesis. A combination of degenerated primers was used for second strand synthesis, followed by tag primed amplification. The mixture of generated amplicons was identified directly by the Fast Sanger sequencing from the anchored 3′ genome end. Evaluating the assay on human serum RNA spiked with viral RNA representing the four dengue serotypes demonstrated a detection limit of 44–124 copies viral RNA per reaction for a two‐step format of the anchored Pan Dengue RT‐PCR and 100–500 copies for a one‐step protocol, respectively. The different serotypes were clearly identified from the generated sequences. Further, the 5‐hr procedure was evaluated and compared to standard real‐time RT‐PCR protocols on acute‐phase serum samples from patients with confirmed dengue infections. This assay demonstrates a strategy for virus detection, which combines nucleic acid amplification adapted for dengue virus RNA with direct and rapid sequencing. It provides a tolerance for new sequence variation and the strategy should be applicable for other RNA viruses. J. Med. Virol. 82:1701–1710, 2010. © 2010 Wiley‐Liss, Inc.     

登革热病毒多重荧光PCR检测及基因分型方法的研究

目的建立一种登革热病毒双靶基因多重荧光PCR检测方法,用于登革热病毒的实验室诊断和基因分型。方法选取登革热病毒Ⅰ-Ⅳ型病毒保守区设计型特异性引物探针和通用型引物探针。评估多重荧光PCR检测方法的特异性、重复性和检测限;并对20份阳性样本进行检测。结果20个登革热阳性核酸标本在通用型检测全部为阳性,特异性型别检测发现登革热病毒Ⅰ型10例、登革热病毒Ⅱ型3例、登革热病毒Ⅲ型3例、登革热病毒Ⅳ型4例;20名正常无症状人群标本提取的核酸和HIV、HCV和HEV通用型和特异性型别检测全部为阴性。梯度检测的变异系数均小于5％。对登革热Ⅰ-Ⅳ型病毒检测最低检测限达10^3 eopies／ml。结论本研究建立的登革热病毒双靶基因多重荧光PCR检测及分型方法具有特异性好、重复性好、快速易操作等优点,可用于登革热病毒的快速检测和基因分型鉴定。     

Rapid detection of common viruses using multi-analyte suspension arrays

A method that uses specific oligonucleotide probes coupled to a specific array of fluorescent microspheres in multi-analyte suspension arrays was employed for the detection of common viruses, such as Herpes virus (HSV), Human papillomavirus (HPV) and Hepatitis B virus (HBV). Sixteen species-specific probes and 9 sets of specific primers were designed based on conserved sequences of these viruses in the GenBank database. Serial symmetric PCR, asymmetric PCR and multiple PCR assays were employed to evaluate the sensitivity, specificity and reproducibility of multi-analyte suspension arrays analyzed on a Luminex-100 analyzer instrument. The symmetric PCR amplification of four types of HSV, four types of HPV and HBV genotypes of B, C and D, combined with their corresponding species-specific probes and specificities were completely concordant with the results from a comparative sequence analyses. There was no significant difference in the median fluorescence intensity (MFI) value between symmetric PCR and asymmetric PCR when the viral DNA concentration was above 10⁴ copies/test. Both PCR products were negative in the multi-analyte suspension arrays with viral DNA concentrations less than 10³ copies/test. A multi-analyte suspension array is a flexible, high-throughput, relatively simple method for rapid identification of common viruses in the clinical laboratory.     

Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.     

Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.