Development Of Real-time PCR For Detection Of Dengue Virus Type1

1. Background and ObjectiveDengue virus belongs to a member of Flavivirus. It is an enveloped single stranded positive sense RNA virus. There are four serotypes of DENV, DENV1to DENV4, which are transmitted to humans through the bites of mosquito and can cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue viruses are transmitted in tropical and subtropical countries that circle the globe. Yearly, an estimated80-100million cases of dengue fever and about250000-500000cases of dengue hemorrhagic fever/dengue shock syndrome occur worldwide. Up to now, there is no effective measure to prevent and control dengue infections, and no vaccine is commercially available. Dengue virus is a wide distribution, incidence, the more harmful to human infectious diseases, and has become the focus of global public health.Laboratory diagnosis of DENV infection is based on virus isolation, detection of virus antigen or detection of DENV specific antibodies. Serological diagnosis is, however, cross-reactive to other flaviviruses. Virus isolation, since time-consuming, high technical requirements, is unsuitable for clinical use. The development of molecular detection offers the possibility of early diagnosis. Application of ordinary RT-PCR method can be performed within1day of dengue virus identification and differential diagnosis. But the method is easy to cause the cross contamination between specimens and result to the false-positive results. Most recently, real-time PCR has been developed to detect DENV in human samples. Real-time PCR technique, compared with conventional PCR technology, has higher sensitivity, specificity and reproducibility, and also has rapid, high throughput, low pollution characteristics. This technique is an important improvement for the rapid diagnosis of DENV infection allowing early initiation of patient management and specific preventive health measures. We developed one system of real-time PCR for the specific detection of dengue virus typel. This diagnosis method was validated for sensitivity, specificity, linearity and precision (repeatability and intermediate precision) on RNA transcripts.With the rapid development of molecular biology technology, real-time fluorescent quantitative PCR is more and more extensive in biological research. Real time fluorescent quantitative PCR technique is defined that fluorescent group was added in PCR reaction system, using fluorescence signal monitoring of the entire process of PCR, and using the standard curve analyzing unknown template. Real-time PCR add fluorescent dye or fluorescent probe in the basic of conventional PCR. Fluorescent dyes can specially incorporate to DNA double strand and emit fluorescent. But without the incorporation of double chain of the dyes do not emit fluorescent signal. So it can ensure that the fluorescence signal increases along with the increase of PCR product. Fluorescence probe method is that the fluorescence resonance energy transfer technology is used in conventional PCR, in the5’ end of probe labeled with a fluorescent reporter group (R),3’end labeled with a quenching group (Q), both of which can be composed of an energy transfer structure, namely the fluorescence emitted by the5’ end can be absorbed or inhibited by quenching group. When the distance of the both is far, inhibition disappears, and the fluorescence signal of reporting group enhances, so the fluorescence monitoring system can receive the fluorescence signal. As the above fluorescence principle, fluorescence signal changes can be continuously detected in the reaction system. When the signal is enhanced to a certain threshold (PCR responses in the anterior15cycles of fluorescence signal as the fluorescence background signal, the field value defaults to the10times of the standard deviation of3-15circular fluorescent signal), Ct value is recorded. C stands for Cycle, T represents threshold, Ct value meaning is: the cycle number when each reaction tube fluorescent signal reaches the set domain value. In the process of this development, two important discoveries play a crucial role:①In the early1990s, the discovery of Taq DNA polymerase, which can degrade the fluorescently labeled probe, make the indirectly detection of PCR products become possible.②Thereafter, the use of fluorescent double labeling probe make the real time monitor of the whole process in a sealed reaction tube become possible. The combination of the two discoveries and the development of instruments and reagents led to the wide use of real time PCR. In1996, ABIPrism7700and5700of the America Applied Biosystems (ABI), LightCyclerTM of Roche have pioneered the commercial use. The amplification and detection are full automatically, short timely, high effectively. The domestic uses ABI Prism7000,7900,7700and LightCyclerTM more. The Q-PCR instrument of ABI Prism7900can be used for high throughput (384plates), while ABI Prism7000using multiple channel technology. Currently the most commonly used real time fluorescence quantitative PCR product inspection technology is the application of fluorescent dyes or group. And PCR and real time signal detection are combined. The most simple one is double-strand DNA chimeric fluorescent dye technology. This method has low cost. But the SYBR Green fluorescent dye can combine with all of the DNA double chain, so the specificity is worse than Taqman probe method. In order to use fluorescent dye.method to get better quantitative results, the specific design of PCR primer and the quality of PCR response require relatively high. While the Taqman probe method is highly specific quantitative PCR technology. Its core is using Taq Enzyme’s3’-5’exonuclease activity to cut off the probe and generate a fluorescent signal. Because the binding of probe and the template is specific, the fluorescence signal intensity is on behalf of the number of templates. With the in-depth study and reagent commercialization, there are more fluorescent quantitative PCR detection technology, such as taqman MGB method, Molecular Beacon method, Amplisensor, Lightcycler, complex probes, as all.2. Methods1) Primer probe design and the establishment of stand curve:80complete sequences of DENV1, each10complete sequences of DENV2,3,4and other flavivirus were downloaded from GenBank, and were aligned with ClustalW to choose specific primers and probes by Beacon Designer7.0software. We diluted the plasmid which contained the conserved sequences10times serially.2) Specificity, repeatability, reproducibility, sensitivity of the real time PCR:we used the real time PCR method to detect the standard strains of dengue virus type1-4and the live attenuated Japanese encephalitis vaccine strains. The linearized plasmid was diluted from1.01×107to1.01×102copies/μl. Different time repeated three times and each time to do three repetitions. In addition, the PMDns2a plasmid and the RNA were10fold diluted to compare the sensitivity.3) Detection of clinical specimens:the cDNA provided by the Eighth People Hospital of Guangzhou, and the serum of suspected patient from Dongguan were detected by the real-time PCR. We used the general primers and probes for verification. We compared the real time PCR and the conventional PCR according to the Ct values.3. Results1) The establishment of stand curve:we got the correlation coefficient of the standard curve was R2=0.999, amplification efficiency was Eff=96.5%, regression equation was Y=-3.4101ogX+34.87(Y as Ct, X as the concentration of template). We did the correlation and regression analysis, the linear correlation coefficient r=-1.000, P=0.000, the regression coefficient is-3.410, P=0.000. So the standard curves of Ct value and initial concentration has a good linear relationship.2) Specificity of the real time PCR:The detection of dengue virus type1was positive, but the detections of the other three types of dengue virus and Japanese encephalitis were negative.3) Repeatability and reproducibility:Repeatability standard deviation (SDr) was between0.04and0.44, reproducibility standard deviation (SDR) was between0.14and0.29. The coefficient of variation was lower than2%. Through the factorial analysis of variance, the F value of the concentration was10528.497, P=0.000, while the F value of the time is0.451, P=0.0.641. It proved that the results of same concentration but different time had no statistical difference, so the method has good repeatability and reproducibility.4) The sensitivity of the real time PCR:the sensitivity of the plasmid can reach to lcopy/μl, and the RNA can reach to100copies/μl. We did the correlation and regression analysis of the Ct value and RNA concentration. The linear correlation coefficient r=-1.000, P=0.000, the regression coefficient is-3.305, P=0.000. So we got the regression equation Y=-3.3051ogX+39.727(Y Ct value, X RNA concentration).5) The detection of clinical specimens:the positive rate of the real time PCR was significantly higher than that of conventional PCR method.4. ConclusionWe succeed to establish a method that can be used to detect the dengue virus type1. The real time PCR may realize to the fast detection and differential diagnosis of the dengue virus type1. This method has the advantages of fast, sensitive, specific and can quantitate the virus samples.