DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

Abstract

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.

Infectious diseases detection by microarray: An overview of clinical relevant infections

“…Derived from the enormous number of viral agents that can cause HF, some molecular methods have been used for diagnostic, for instance rtPCR and real time PCR [26]. Based on microarrays, a detection and identification approach was designed for seven agents of the Flaviviridae family: yellow fever, West Nile virus (WNV), Japanese encephalitis, and the dengue 1 -4 viruses, which are causing severe human disease in tropical and subtropical areas all over the world [27].…”

Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus

“…It also may allow for the detection of those mutations even when they occur at relatively low levels (up to 1%) as in the case of mixtures of quasispecies that cannot be detected by traditional direct sequencing methods (Cherkasova et al, 2003;Leberre at al., 2007). The efficiency of microarrays for identification and discrimination of closely related bacteria and viruses has been previously demonstrated Hsia et al, 2007;Laassri et al, 2003;Nordström et al, 2005;Volokhov et al, 2002;Wade et al, 2004). The use of oligonucleotide microchips for screening of random mutations is based on the ability of microarrays to identify the presence of singlenucleotide mutations in the hybridization template (Hacia et al, 1999;Urakawa et al, 2003).…”

Anchored Pan Dengue RT‐PCR and Fast Sanger sequencing for detection of Dengue RNA in human serum

“…For reference, a protocol used widely for random hexamer 18 cDNA synthesis produced up to 8 times more cDNA than the developed 3'RT protocol. The 19 higher amount of cDNA produced for the random hexamer protocol was probably due to both 20 cDNA synthesis from viral RNA lacking the outermost part of the 3' genome end, which was 21 not targeted by the 3'RT-p1 primer, as well as a higher stringency for the 3'RT protocol. 22…”

“…A combination of degenerated primers was used for second strand synthesis, providing 18 limited dengue virus specificity and thereby some tolerance for new sequence variation. The 19 following amplification targeted a mutual tag primer on the 5' end of the primers used for 20 cDNA and second strand synthesis, both to facilitate the application of highly degenerated 21 second strand primers as well as to find the optimal amplification conditions for a 22 combination of several primers. Fast Sanger sequencing was carried out on the mixture of 23 generated amplicons from the anchored 3' genome end using the cDNA primer.…”