Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.     

Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes

A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 x 10(2) to 3 x 10(3) copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7-8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock.     

双重荧光定量RT-PCR快速检测流感病毒H1、H3亚型的研究

目的利用荧光定量RT-PCR技术建立一种快速检测流感病毒H1、H3亚型的方法。方法根据H1、H3亚型流感病毒HA基因的相对保守序列,设计两对引物及其相应的Taqman探针,利用一步法RT-PCR试剂盒建立优化反应体系后,将荧光定量RT-PCR的产物采用10倍稀释法,即107～100copies/μl,再次用荧光定量PCR方法检验建立体系的灵敏度和重复性,并建立相对定量标准曲线;利用多种流感病毒和具有相似临床症状的呼吸道病毒检验建立体系的特异性。结果 H1和H3亚型流感病毒的检测灵敏度为102copies/μl,扩增效率分别为101.35%和113.28%,标准曲线相关系数大于99%,重复性良好,特异度实验未发现有非特异性扩增。结论本研究建立的双重荧光定量RT-PCR技术可以快速、准确地检测H1、H3亚型流感病毒。     

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.     

Development of a real-time reverse transcription polymerase chain reaction assay for detection of marine caliciviruses (genus Vesivirus)

Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses.     

Development of real-time RT-PCR for detection of human metapneumovirus and genetic analysis of circulating strains (2009-2011) in Pune,India

Human metapneumovirus (HMPV) is an important respiratory virus implicated in respiratory infections. The purpose of this study was to develop a one-step real-time RT-PCR assay that can detect all four lineages of HMPV and to identify the HMPV lineages circulating in Pune, India. Conserved regions of the nucleoprotein gene were used to design real-time primers and a probe. A total of 224 clinical samples that were positive for different respiratory viruses (including 51 samples that were positive for HMPV) were tested using the real time RT-PCR assay, and the specificity of the assay was observed to be 100 %. Using in vitro-synthesized RNA, the sensitivity of the assay was ascertained to be 100 copies of the target gene per reaction. Phylogenetic analysis of the nucleoprotein (N) and attachment glycoprotein (G) genes confirmed that this assay detected all lineages of HMPV. A2, B1 and B2 strains were observed during the study period. Our assay is highly sensitive and specific for all known lineages of HMPV, making it a valuable tool for rapid detection of the virus. A2 and B2 were the predominant subtypes circulating in Pune, Western India.     

Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens

Real-time RT-PCR assays targeting sequences in the measles virus (MV) nucleoprotein (N), fusion (F), and hemagglutinin (H) genes were developed for the detection of MV RNA in clinical specimens. Four primer and probe sets each for the N, F, and H genes were evaluated and reaction conditions optimized. Using dilution series of synthetic RNAs, the limits of detection were determined to be approximately 10 copies for each target RNA/reaction. The relationship between C(t) values and RNA concentration was linear within a range of 10-10(6) RNA copies/reaction, and intra- and inter-assay variability was low. The N gene-specific real-time assay detected MV RNA in 100% of clinical samples from confirmed measles cases compared to 41% by standard RT-PCR. The MV H and F gene-specific real-time assays detected MV RNA in 93% and 82% of these specimens, respectively. Real-time assays could detect RNA from strains representing each active genotype of MV and were also highly specific, as no false positives were identified when samples known to contain other respiratory viruses were tested. Real-time RT-PCR assays will be available to support routine measles laboratory surveillance, to facilitate research projects on pathogenesis that require sensitive and quantitative detection of MV RNA, and to aid in the investigation of serious disease sequelae resulting from natural measles infection or vaccination with measles-containing vaccines.     

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.     

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.