In this study, we have eastablished a real-time quantitative reverse transcription polymerase chain reaction assay(qRT-PCR) based on DEV UL55 gene successfully. It is a powerful tool for quantitative analysis of target RNA due to its high-throughput, together with excellent sensitivity and specificity, low contamination risk, ease of performance and speed, more reliable instrumentation and improved protocols. The application of it in quantitative analysis of DEV UL55 gene samples has demonstrated the transcription of UL55 gene occures in the last stage of viral replication cycle.
Reverse transcription combined with real time PCR has been created as qRT-PCR, which has become the standard for the detection and quantification of RNA targets and firmly established as a mainstream research technology due to the outstanding advantages. To date, the technology has been used vastly in fields of pathogeny detection(virus, bacterium, fungus, etc), gene expression analysis(cell factor, growth factor, transcription factor, etc) and so on[28, 29]. To our knowledge, viruses complete their composition in host cell through the expression and replication of viral genome. Gene expression mechanisms of them in a cell act as both an "on/off switch" to direct which genes are expressed, and as a "volume control" in regulating gene expression. The monitoring of gene expression will be a useful maker of disease progression and as a component of studies into the efficacy of antiviral compounds. Real time quantitative PCR became an attractive method to study gene expression because of its low inter-assay and intra-assay variablilty and its equivalent or greater analytical sensitivity in comparison with traditional method. The quantification of templates by qRT-PCR can be performed in two ways: as absolute quantification and as relative quantification. The former should be performed in situations where it is necessary to determine the exact transcript copy number of targets present in the sample. Nevertheless, the latter describes the change in expression of the target gene relative to some reference group such as an untreated control or a sample at time zero in a time-course study. Thus, we employed relative quantification as an ideal method to study dynamic transcription profiling of DEV UL55 gene in this paper.
In theory, qRT-PCR differs from PCR mainly by the addition of a preliminary step, the initial conversion of RNA into a DNA template by an RNA-dependent DNA polymerase(reverse transcriptase). In fact, this additional procedure results in a much more fragile and variable assay[10, 32]. Expression levels of target genes measured by real-time RT-PCR should be normalized by comparison with transcript abundances of reference genes to.avoid serious distortion of experimental results. β-actin occurs in all duck nucleated cell types would be universally valid due to a constant expression level regardless of the expremental conditions[34, 35]. Results demonstrated the introduction of it has corrected the sample-to-sample variation of UL55 which might occur at a number of stages throughout the experimental protocol of RNA quantification experiments and affect efficiencies of the reverse transcription(RT) and polymerase chain reaction(PCR) reactions. Besides, mock infected DEFs were introduced as homologous controls for alternative normalization. They were amplified with UL55 gene using the same PCR primers and have eliminated the introduced contamination during our experiment as we expected.
The principle of qRT-PCR assays is straightforward: following the RT of RNA into cDNA, it requires a suitable detection chemistry to report the presence of PCR products, an instrument to monitor the amplification in real time and appropriate software for quantitative analysis. Detection chemistries can be either probe- or non-probe based. Although the specificity of non-probe based chemistries depends on the specificity of the primers, an important advantage of coverting the optimized conventional RT-PCR assays immediately into real-time assays has made it to be an ideal method for quantification. The most widely used non-probe-based chemistry detects the binding of SYBR Green I to ds(double-stranded) DNA. SYBR Green binds to the minor groove of dsDNA, causing fluorescence signals to increase synchronously during PCR as DNA products increase exponentially. Using SYBR Green I for quantification of DEV UL55 gene has eliminated the need for complicated probe design. However, optimization of PCR conditions was supposed to be carried out prior to the use of SYBR Green I to reduce the effect of the nonspecific binding to PCR artifacts such as primer dimers that may contribute to the fluorescence signal. The single band generated by coventional PCR as we expected has demonstrated the the primers we designed for UL55 gene and β-atin were specific and availabe for amplification. Also, melting curve analysis of UL55 gene and β-atin comfirmed the conclusion of primers as the single peak of each curve presented in our research. Meanwhile, the optimized protocol was determined by the way. All these ensured the protocol has a low potential to form secondary structures, including self and crosshybridization with other oligonucleotides in the PCR.
Quantification can be relative to an external standard curve based on the use of a dilution series of an external standard, which can be used to generate a standard curve of Ct(threshold cycle) against initial target copy number. In our reaserch, the plasmids pMD18-T/UL55 and pMD18-T/β-actin were particularly constructed to generate standard curves. The copy numbers of unknown samples can be calculated from the linear regression of that standard curve, with the log(nanograms) input amount of RNA in each well as the x values and Ct cycle as the y values. The most important parameters Ct was defined as the cycle when sample fluorescence exceeds a chosen threshold above calculated background fluorescence. The determination of it depends upon the sensitivity and ability of the instrument to discriminate specific fluorescence from background noise, the concentration and nature of the fluorescence-generating component and the amount of template initially present. The more templates present at the beginning of the reaction, the fewer cycles it takes to reach a point in which the fluorescent signal is first recorded as statistically significant above background. Since background fluorescence is not a constant or absolute value but is influenced by changing reaction conditions, the value of a Ct recorded for a particular sample might vary with the variation of background fluorescence varies. In order to avoid false positive results, we runned samples in triplicate with NTC(no templates control) as negative control which is too low to cross the default threshold level. If the corrections results of a negative control becoming positive, it makes the result unbelievable. Moreover, it has been reported that the NTC recording a Ct less than 30 suggested the presence of high levels of contamination in the laboratory. After caculation, the standard curves of pMD18-T/UL55 and pMD18-T/β-actin were described by the equation: Y = -3.321X + 7.755 and Y = -3.279 + 6.465, respectively. The Ct value and the correlation of established standard curves demonstrated an excellent linear relationship of them, and the method we established can be used for the detection of UL55 gene in an extensive boundary. In addition, the amplification efficiencies of pMD18-T/UL55(100%) and reference pMD18-T/β-actin(101.8%) were quite close to each other. It was crucial because it affects the accuracy of any calculated expression result directly[11, 41].
It has been reported that the transcription of herpesvirus genes were processed by stages: the expression of immediate-early(IE) genes occured first, following by the expression of early(E) genes and late(L) genes sequentially. In order to figure out the type of DEV UL55 gene and the transcription profiling of it, as well as how it changes over time, we collected triplicate samples of cells at different time. The dynamic proliferation of UL55 gene calculated by iCycler IQ 5 suggested the transcription of DEV UL55 gene became detectable at 8 h p.i. compare to negative control, peaked at 36 h, then decreased but kept a high level until 60 h p.i. etc. According to the gene expression time series terminology, DEV UL55 gene could be clustered to late genes, which was consistent with the report of HSV-1, HSV-2 UL55 gene. That probably means the product of DEV UL55 gene may execute the similary function of late genes in DEV life cycle such as HSV UL55 gene did. It has been reported that late genes predominantly encode proteins to form the virion particle. Packaging of the viral particles including viral assembly, maturation, egress, and release which mostly occurred at the later stages of DEV life. DEV UL55 gene started transcription late in host cells and kept a comparatively high leve during our detection can be the resonable interpretation of UL55 gene production participating in the above procedures.
Late genes are subdivided into two categories as leaky-late(γ1) or strictlate(γ2). The γ1 genes can be suboptimally expressed in the absence of viral DNA synthesis, whereas the γ2, have a strict requirement for viral DNA synthesis. Thus, we dealt the infected DEF cells with some canonical medicine to analyze a temporal regulation condition for UL55 gene. Studies of vulnerable patient populations of HSV have indicated that daily use of antivirals such as acyclovir and valacyclovir can reduce reactivation rates due to the interference with viral replication. Evaluation of effection of antivirus drug used for DEV in vitro proved acyclovir had a good inhibitory activity against DEV. Based on the unavailable phosphonoacetate and acyclovir in reality, we replaced it with ganciclovir as an alternative which has the same inhibition for DNA synthesis. Results in our study indicated DEV UL55 gene was sensitive to ganciclovir since the synthesis procedures of DNA have been inhibited by corresponding inhibitor. In other words, that implied DEV UL55 gene actually is a γ2 gene whose transcription was mainly depends on the DNA which has been synthesized previously.