P Somvanshi, P Seth
dengue, primers, structural region
P Somvanshi, P Seth. Insilico primer designing of structural region of Dengue virus for molecular diagnostic. The Internet Journal of Genomics and Proteomics. 2007 Volume 3 Number 2.
Dengue has reemerged significantly from the past two decades for the public health problems. Clinicians require a diagnostic test with high sensitivity, which is applicable during early symptomatic phase. PCR is a specific and sensitive molecular technique to detect the gene for monitoring possible Dengue outbreaks and circulating viral serotypes. Bioinformatics tools were used to design and verify the novel Dengue subtype specific primers. In this investigation, 24 primers targeting the structural region of four variants of dengue virus were designed through
Dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are a major public health problem globally found mostly in tropical and subtropical countries (Rigau-Pérez et al 1994; Gubler, 1998). These infections are of considerable public health importance because of the high morbidity and mortality associated with their worldwide distribution. The known geographic range of dengue includes the Americas, Africa, Asia and the South Pacific determined by the distribution of its mosquito vectors. Movement of dengue in about 2/5 of the world population among different geographic area is an important element in the epidemiology of the disease (WHO, 2002). During a Dengue virus epidemic, infection rates among those susceptible to the virus are generally 40-50% but could potentially reaches as high as 80-90%.It is estimated that 5 lakh cases of DHF require hospitalization and a minimum of 2.5% of them resulted in death. The fatality rate of DHF, if untreated, is around 20% but proper treatment could reduce them to about 1%.At present it is estimated that each continent has 5% fatality rate for those infected with Den V, with children and young adults being the groups which are most susceptible to fatalities from infection.(WHO, 2002).
The dengue virus is a single-stranded positive-strand RNA virus. The four serotypes of dengue virus exists viz DEV I-IV. The genome size of all four dengue virus was approximately 11kb in length (Halstead
The current strategy was prepared to identify and characterize the dengue virus in the laboratory through the cell culture technique. However, this requires cell culture facilities which are difficult to maintain in laboratories (Harris et al 1998). A viral growth usually takes long time and always in the risk of contamination. Two different methods have been used in dengue diagnostics, immunological ELISA and PCR. Polymerase chain reaction is a sensitive, specific and rapid molecular assay to identify and characterize the virus. The dengue suspected person blood sample was extracted and isolation of the RNA was done and it was converted into the cDNA. The cDNA will be used as a template for specific molecular diagnostic of dengue viruses. The detection of RNA virus with molecular technique viz reverse transcriptase polymerase chain reaction (RT-PCR) (Alcon
Bioinformatics provide an innovative strategy to design and validate the primers. Currently, several
Material And Methods
Collection of sequences
The complete nucleotide sequences of structural region of envelope, capsid and premembrane of Dengue virus I, II, III, IV were extracted from biological database, viz. National Centre for Biotechnology Information (NCBI) cited at http://www.ncbi.nlm.nih.gov
Strategies for designing of PCR primers
The open reading frame of structural gene was prepared from the whole genome of dengue virus. These nucleotide sequences
Basic Local Alignment Search Tools (BLAST)
The relatedness of sequences deposited in databases was evaluated by BLAST (Basic Local Alignment Search Tool), (Altschul et al., 1990) implemented via the NCBI website [www.ncbi.nlm.nih.gov/blast/] against the complete training dataset which is extracted from Genbank database. The Blastn (nucleotide query – nucleotide database comparison) in which conditional composition score adjustment having no filters of BLOSUM 62 matrix with threshold expect value 10 were used. Specificity and cross reactivity was check by PSI-BLAST.
Results And Discussion
Total four serotype of known dengue virus used in the study was given (
The total genome size of dengue studied was about 10 kb and encodes the structural and non structural region. Structural region of genome are helpful in the coating of capsid, envelope and membrane glycoprotein. These regions are essential for the virus replication and helpful in the infection in the hosts. The structural regions are very important for the molecular based diagnostic of dengue virus. In this investigation, all the three important structural region of dengue virus have been used to design the dengue specific primers. The primers were also verified for their cross reactivity with other organisms, resultant there was no cross reactivity observed within the dengue virus or other virus. The GC contents, melting temperature, expected size of PCR product and position of primers pair in the genome of dengue virus was given (
The primers might be suitable for the mutagenesis of dengue virus. These primers may be helpful for PCR amplification complete structural gene and also for the expression of all three regions. The expressed protein might help in the immunodiagnostic of all four dengue virus. The non structural region of DNA is helpful in the stability of the genome and virus. These non structural regions may be used in the taxonomy and classification of dengue virus. In our previous study, the conserved domains present in the genome of
There was no cross reactivity observed in the primers within the dengue virus and other organisms. As the NCBI BLASTN is an online program use for the homology search. In this study, all these primers were confirmed with this programme. There was no primers homology match with the other organisms. Dengue I primers were found matching with the dengue I sequences database. And other dengue II, III and IV were harmonized with the II, III and IV respectively. Therefore, we can state primers are specific and does not cross react with others organisms. In this study, different set of PCR primers have been used to identify the dengue. The molecular techniques are able to directly assess the presence of viral RNA in plasma/serum samples from subjects with suspected disease. Actually, reverse transcriptase PCR was found extremely practical in the recent outbreak of severe acute respiratory syndrome, when medical staff could quickly avoid the adoption of quarantine measures for dengue RNA-positive patients (Barkham et al 2006). Several RT-PCR methods for dengue RNA detection includes both conventional and real-time PCR have been described (Norman et al 2001).
In conclusion, we report the development of specific primers for molecular level diagnostic of structural region of dengue viruses. The primers may be a support for the diagnostic of dengue and dengue hemorrhagic infection in humans. PCR based techniques will be useful for early molecular detection and control of Dengue viruses and in this way it provide the support for human health management.