NUEVA S/E SECCIONADORA BULI .1 Situación existente

Microarray technique has emerged as a new functional tool in genomic research and clinical laboratories (Peytavi et al., 2005). Figure 1-6 clarifies the principle of this nanoscale technique. It includes use of synthesized fragments of recombinant nucleic acids or oligonucleotides (probes) affixed on a specific chip which is either a nylon membrane or the surface of a glass slide. These probes are able to hybridize with complementary nucleic acid sequences.

Fluorescent labelling dyes (e.g. platinum compounds) can facilitate the detection of this type of hybridization (Shieh and Li, 2004). Based on its high capacity to accommodate hundreds to thousands of individual viral gene probes and allow simultaneous detection of any amplifiable pathogen present in a specimen, microarray technology is able to overcome the limitations of the conventional methods which are applied for the diagnoses of viral infections which are virus or pathogen specific.

Figure ‎1-6: Shows the schematic drawing of multivirus chip.

This technique has certain advantages over the conventional methods used for detection and identification of the microbial pathogens, which they are:

1- The complication rates of false positive results due to contamination are highly limited.

2- The microarray chip can be designed with high flexibility to detect all of the viral pathogens. It can be also rapidly modified to match the emergence of new viruses.

3- Viral gene expression as well as genotyping of viral isolates can be achieved by this process (Shieh and Li, 2004).

4- Many viruses can be identified by microarray at the same time. Therefore, it is highly recommended for the diagnosis of specimens which are suspected to contain multiple pathogenic viruses (Elnifro et al., 2000).

This technique has been employed together with PCR amplification for the simultaneous identification of several pathogens in only one process (Miller and Tang, 2009). A microarray viral system was firstly described by (Wang et al., 2002) aiming to identify the pathogens of severe viral respiratory infections. This chip contained about 1600 probes covering 140 viral genomes including the serotypes of RSV, parainfluenza, adenoviruses and human rhinoviruses. This was later developed into a pan-viral microarray by using approximately 10,000 highly conserved sequences of 70-mer oligonucleotides representing the full reference sequence of 1000 viral genomes from GeneBank in order to improve the identification possibility of both known and unknown members through cross-hybridization to this array of specific probes. A modified random PCR protocol was used to amplify viral genomes in clinical specimens. This procedure starts with the synthesis of first- and second strand

by using specific primer-A (5’- GTTTCCCAGTCACGATCNNNNNNNNN), this will be completed with 40 cycles of PCR amplification process with another specific primer-B (5’-GTTTCCCAGTCACGATC). Generally, this protocol includes three rounds of PCR (A, B and C). In round A, cDNA is synthesized using primer A. In round B, the generated templates (first and second strand cDNA) are then amplified using primer B. The amplification is completed in round C through incorporation of an additional group, which is either an amino allyl deoxyuridine triphosphate (dUTP) or a cyanine-dye- coupled nucleotide, using additional PCR cycles. More additional details are found at http://dx.doi.org/10.1371/journal.pbio.0000002.sd002 (Wang et al., 2003). The virochip was applied globally to discover the virus associated with SARS (Ksiazek et al., 2003).

DNA microarray technology was devised by Boriskin and his group in 2004 for the detection of viral neuropathogens (e.g. VZV, HSV and enteroviruses) in persons with encephalitis and meningitis especially those very young and immunocompromised patients. Multiplex PCR was used to amplify viral genetic materials in clinical samples especially those with low quantities such as CSF. This array was able to identify up to 13 different viruses in one panel. The Affymetrix GeneChip system was designed by (Malanoski et al., 2006) for the instantaneous identification of respiratory pathogens through resequencing the short oligonucleotides of the original platform which aided in the identification of new subtypes of influenza virus H5N1 and H1N1 in 2007 and 2009, respectively. GreenChipResp platform was designed later by (Quan et al., 2007) and utilized for the detection of subtypes of influenza-A viruses and other respiratory viruses, and was then developed for the identification of the

coronaviruses (Miller and Tang, 2009). MChips was created by (Dawson et al., 2006) and employed for the identification of the influenza-A in recent and historical specimens through the detection of the polymorphisms in their M gene. This MChip was also used for the identification of influenza-B in similar samples and those subtypes of both influenza A and B (H1N1, H3N2 and H5N1) which infect humans (Mikhailovich et al., 2008).

Simultaneous identification of seven human herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6A and HHV-6B) in a single reaction was achieved by (Zheng et al., 2008) based on the conserved regions of DNA polymerase gene of the previous viruses which are used to develop a cheap DNA microarray chip.

Microarray technology was used as a novel approach to identify and quantify hepatitis B and C; and Human immunodeficiency virus type 1 (HIV-1) in the plasma specimens of donors (Khodakov et al., 2008).

Amplification process is necessary in microarray technology in order to amplify the minute amounts of viral genomes in clinical samples. PCR is the common method used for exponential amplification of nucleic acids, but this method can only generate fragments with maximum length 500 base pairs. Degenerate oligonucleotide primed-PCR (DOP-PCR) represents the recent technique used to amplify the entire genomes of different organisms from any source through using partially degenerate primers (primers containing few of the degenerate nucleotides shown in table 2-9) which can facilitate binding these primers to their complementary nucleic acid sequences in a PCR protocol with two different annealing temperatures (Fortina et al., 2001).

The successful isolation of the isothermal Phi 29 DNA polymerase from Bacillus subtilis bacteriophage Phi 29 which has the ability to replicate the target nucleic acid isothermally, opened the door for continuous nucleic acids amplification along the entire microbial genome using random hexamer primers in a productive isothermal process called whole genome amplification (WGA).

In this process, some molecules of the Phi 29 DNA polymerase start elongation of random hexamers annealed to the complementary sequences of the target denatured DNA without disassociation from the original template. At the same time, other molecules of the Phi-29 DNA polymerase partially displace the beginning of the new extending complementary strands, which provides new templates ready for more isothermal multiple displacement amplification (MDA) reactions. Continuous priming and strand displacement led to exponential amplification and generated high molecular weight of hyperbranched amplicons containing various lengths of linear dsDNA, ssDNA and some forks of nucleic acids. Therefore, MDA is being used as an appropriate method for WGA of nucleic acids in the absence of prior knowledge of organisms that may be present at a rate of more than (104) copies/ml (figure 1-7) (Lage et al., 2003, Monstein et al., 2005, Lovmar and Syvanen, 2006, Binga et al., 2008, Maragh et al., 2008, Wang et al., 2011, Zheng et al., 2011).

Figure ‎1-7: Schematic diagram shows the working principle of Phi 29 DNA polymerase during MDA process.

In figure A, the random hexamers primers represented by the short green lines anneal to the single strand DNA represented by the long blue line. In figure B, the Phi 29 DNA polymerase molecules represented by the black circles start extending the annealed random hexamers primers till reaching the newly synthesized complementary strands of DNA represented by the orange lines. In figure C, the Phi 29 DNA polymerase molecules displace the newly synthesized strands and continue the polymerization, at the same time more primers of random hexamers annealed to the newly synthesized complementary strand of DNA. In figure D, polymerization starts on the new strands, forming a hyperbranched structure. The black arrows show the direction of the new strands synthesis.

Adopted from (Spits et al., 2006).

In general, it has emerged as an ideal method for the extensive parallel identification and differentiation of various pathogens and their strains in clinical samples (Maughan et al., 2001).