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Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene.

ABSTRACT: BACKGROUND: Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet. RESULTS: The newly identified duck enteritis virus UL55 gene was amplified and cloned into pMD18-T vector after digestion to generate a recombinant plasmid pMD18-T/UL55 for the establishment of qRT-PCR as standard DNA. The results of agarose gel electrophoresis and melting curve analysis demonstrated the primers we designed for qRT-PCR were specific and available. We used ?-actin as a reference gene for normalization and established two standard curves based on pMD18-T/UL55 and pMD18-T/?-actin successfully. Based on that, the transcriptional analysis of DEV UL55 gene was performed, and the result suggested the expression of UL55 mRNA was at a low level from 0 to 8 h post-infection(p.i.), then accumulated quickly since 12 h p.i. and peaked at 36 h p.i., it can be detected till 60 h p.i.. Nucleic acid inhibition test was carried out for analyzing a temporal regulation condition of DEV UL55 gene, result revealed that it was sensitive to ganciclovir. Synthesis procedures of DEV UL55 gene can be inhibited by ganciclovir. CONCLUSIONS: The method we established in this paper can provide quantitative values reflecting the amounts of measured mRNA in samples. It's available for detection and quantification, also can be used in DEV diagnosis. The DEV UL55 gene was produced most abundantly during the late phase of replication in DEV-infected cells and the transcription of it depended on the synthesized DNA. DEV UL55 gene is a ?2 gene which occurs last and have a strict requirement for viral DNA synthesis.

SUBMITTER: Wu Y

PROVIDER: S-EPMC3120788 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene.

Wu Ying Y Cheng Anchun A Wang Mingshu M Zhang Shunchuan S Zhu Dekang D Jia Renyong R Luo Qihui Q Chen Zhengli Z Chen Xiaoyue X

Virology journal 20110601

<h4>Background</h4>Real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) has become the benchmark for detection and quantification of target gene expression level and been utilized increasingly in detection of viral load and therapy monitoring. The dynamic transcription variation of duck enteritis virus UL55 gene during the life cycle of duck enteritis virus in infected cells has not been reported yet.<h4>Results</h4>The newly identified duck enteritis virus UL55 ...[more]

PMID: 21631934

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Project description:The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus surveillance.

Project description:BACKGROUND AND OBJECTIVES:The variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera. METHODS:The conserved sequences from 5' UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 102 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases. RESULTS:The results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays. CONCLUSION:The multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

Dataset Information

Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene.

Publications

Establishment of real-time quantitative reverse transcription polymerase chain reaction assay for transcriptional analysis of duck enteritis virus UL55 gene.

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