Development Of A Multiplex Real-time RT-PCR Assay With An Internal Control For Detection Of Dengue And Chikungunya Viruses

Backgrounds:Dengue haemorrhagic fever (DHF) and chikungunya fever are cased by dengue virus and chikungunya virus. Dengue virus belongs to family Flaviviridae, genus Flavivirus. Genome for single strand RNA, approximately 11 Kb. chikungunya virus(CHIKV) belongs to Semliki Forest Virus complex,Togaviridae. Genome long approximately 1.18Kb, a total of about 12000 nudeotides encoding protein polymers. The two virus both transmitted by mosquitoes, principally Ae.aegypti and Aedes albopictus distributed in the region where the temperature is higher and the rain is more, so these two disease outbreak mainly in tropical and subtropical regions. In 2013,Brady and his colleagues applied to mapping method evaluated that nearly 400 million people are infected with dengue fever, two times more than the number that WHO evaluated before. There are more than 125 countries experienced dengue epidemic. In 1952, chikungunya fever first outbreak in newari state, southern Tanzania, now there are more than 60 countries experienced chikungunya fever epidemic. it performances similiar clinical smptoms after those two virus infected, mainly characterized by fever, rash, body aches, headache, muscle and joint pain and other symptoms, even can appear some severe symptom like bleeding and shock, which are life-threatening. There is no dengue and chikungunya virus vaccine,so the early diagnosis is important to prevent disease develop to further progress. Laboratory diagnosis of disease that spread by mosquito is based on virus isolation、serological detection and Molecular biological detection. Virus isolation is the gold standard that detection dengue and chikungunya virus. Common used virus isolation method include adult mosquitoes chest inoculate method、suckling mouse brain inoculate method、Sensitive cell line train separation and so on. But this method has a high demand for lab environment, and the experimental period is long, so it is not suitable for the rapid detection of the virus. Serological tests including hemagglutinin inhibition test and IgM/IgG antibody ELISA detection. hemagglutinin inhibition test has simple operation, good repeatability, but the specificity is low, prone to cross reaction, has been gradually replaced by other experiments. Now with the development of molecular biology techniques, RT-PCR can rapid testing and classification of virus, but as a result of this method is easy to cause the cross contamination between samples, therefore is not recommended. At this stage, the real-time RT-PCR detection technology is widely used in rapid diagnosis and classification, it is base on the RT-PCR techniques, to join the fluorescent marker, by detecting fluorescence signal strength to quantitative the virus. Fluorescent markers is divided into the dye and probe method, Dye method is in PCR reaction system, add excessisve SYBR fluorescent dyes, nonspecific and mixed with small ditch in DNA, emit fluorescent signal, the SYBR dye which is not mixed with small ditch in DNA will not launch any fluorescent signal, thus ensuring the fluorescent signal proportional to the amount of PCR product. Probe method in PCR amplification add a pair of specific primers and a specificity of fluorescent probe, labeled a fluorescent report and a fluorescence quenching reactive in the probe ends respectively. When the probe is complete, the fluorescent signal report group launched absorbed by quenching in groups. In PCR amplification,5’-3’ circumscribed Taq enzyme degrade the probe, made the report and the fluorescence quenching reactive separation, thus the fluorescent signal monitoring system can receive the fluorescence, there is a fluorescent molecules formed when amplify a strand of DNA, realized the fluorescent signal accumulation formation and PCR products completely in sync. This experiment is according to the principle of the probe design, for dengue and chikungunya virus and virus detection and quantitative.Methods:1. The evaluation of Different detection method of dengue virus:432 dengue serum specimens from the patients were suspected with dengue infection cases in a dengue outbreak at FoShan in 2014. For the PT-PCR detection、rapid detection of NS1、NS1 enzyme-linked immune detection、IgG and IgM antibody detection respectively. After statistical analysis, compare the positive rate of different methods at different stages of disease.2. design the Primers and probe and prepare the Plasmid standard:Collect dengue virus type (1-4) and chikungunya virus gene sequences as much as possible from GenBank. Using the 6.12 software BioEdit to make the gene sequence alignment, analysis the conserved sequence of the virus, design Primers and the probe by Primer Express3.0. Purify the aimed gene, connected to T-Vector, transformation into the cells. Coate plate on LB AGAR plate with ampicillin added. Pick colony to sequencing confirmed that the correct insertion fragment. Apply Droplet Digital PCR detect the copies of standard plasmid. Make a Synthetic gene as the internal controls, and design the primer and probe for it.3. The sensitivity and specificity of real-time RT-PCR:making standard plasmid 10 times dilution series, apply the real-time RT-PCR doing the test, then make the standard curve, evaluate the detection limit of the real-time RT-PCR. Choose Japanese encephalitis virus, west Nile virus and yellow fever virus, sindbis virus strain doing the test, assessment the specificity of this detection method.4. Real-time RT-PCR detect the clinical samples:clinical samples include 294 dengue suspected Serum samples and 33 CHIKV suspected Serum samples, every sample doing the Gene sequencing detection and real-time RT-PCR detection, as the Gene sequencing detection the gold standard, To calculate the sensitivity, specific, positive/Negative coincidence rate.Results:1. In the test evaluate Different detection method of dengue virus, the cases of serum samples collected in 0-19 day from the day onset. including 212 men (49.0%), 221 women people (51.0%). Women patients with an average age of 40.69 (1-89).2. Real-time RT-PCR detect the number of positive cases are 321, positive rate is 74.13%, positive number of NS1 rapid detection are 350 (80.8%)、positive number of NS1 enzyme-linked immune detection are 350 (80.8%).these three tests have high positive rate in the acute stage of dengue fever, especially in day 3-9 after onset (positive rate of these three methods are all more than 80% during the day 3-9 after onset). The detection of IgG and IgM find the total positive rate are both low (less than 50%), from the ninth day since onset, IgG enzyme-linked immune detection begin to show a upper trend.3. Have Designed the primer and probe in 3’nonstructural protein coding regions of dengue virus and CDS regions of chikungunya virus. Using the T-A clone method make a Plasmid standard which added the aimed fragment of dengue and chikungunya virus4. Standard curve and the limit of detection:The concentration of Plasmid is 9.2×105copies/ul, the CT value of Plasmid standard has a good linear relationship with the concentration. The standard curve of dengue virus is Y=39.66-3.601×lgX, R2=0.997. The standard curve of chikungunya virus is Y=40.22-3.464lgX, R2=0.999. the limit of detection of these two virus are both 103copies/ml.5. Repeatability and specificity:repeatability test of Dengue virus detection result display that coefficient of variation of plasmid standard CT value of different concentration are 1.59%-3.80%, the plasmid standard CT value in different concentrations have statistical significance (F=354.212, P<0.0001), the plasmid standard CT value in the same concentrations have no statistical significance (F=0.007, P>0.05). Repeatability test of chikungunya virus detection result display that coefficient of variation of plasmid standard CT value of different concentration are 1.59%-3.80%, the plasmid standard CT value in different concentrations have statistical significance (F=958.625, P<0.0001), the plasmid standard CT value in the same concentrations have no statistical significance (F=0.002, P>0.05). Detection of Japanese encephalitis virus, west Nile virus and yellow fever virus, sindbis virus strains, are all negative, the specificity is 100%.6. The evaluation of clinical samples:the sensitivity of dengue virus detection is 100%, specificity is 89.3%, Positive predictive value is 92.1%, negative predictive value is 100%.the sensitivity、 Specificity、Positive/negative predictive value of chikungunya are all 100%.Conclusions:This test do the evaluation of different detection method of dengue virus, guide the laboratory choose the best method to detect the dengue virus in the clinical work. After that, we established a multiplex real-time RT-PCR assay that can detect the dengue virus and chikungunya virus simultaneously, and it can quantitate the virus.