1 Nucleic Acid Based Techniques

Transcript

Hello!! I welcome you all to the third week of the course Detection, Diagnosis and Management of Plant Diseases. In the particular course of this week we will be talking about advanced and molecular techniques used in plant disease diagnosis and we’ll start with nucleic acid-based techniques.

So we all know that nucleic acid-based technique is very sensitive and very specific and that requires that is why it has been used widely in today’s context for detection of plant disease of various origin. Plant pathogens that detects through DNA and RNA are based on overcome the certain diagnostics and pathogen taxonomy that enables a rapid and accurate detection and quantification of plant pathogens. Very important to know that the K. Mullis has received Nobel Prize in 1993 due to his discovery for amplification of nucleic acid sequences using the technology known as polymerase chain reaction. In short we popularly known as PCR. Based on the fidelity of DNA hybridization and replication PCR isused for highly specific detection of fungi, bacteria, viruses and phytoplasma So it’s been a very common and popular technology for using in plant disease diagnosis. PCR technique can provide very high sensitivity and specificity due to the fidelity of DNA amplification.

Success of PCR depends on efficacy of DNA extraction and performance is affected by inhibitors present in the sample assay, polymerase activity, PCR buffer and concentration of deoxynucleoside triphosphate. In addition application of PCR for pathogen detection requires designing of a primer to initiate DNA amplification which could limit the practical applicability of this technique for new and unknown pathogens. If the DNA sequence of the new or unknown pathogen is not available then designing primer will not be possible and that is why this is a limitation of PCR. PCR based methods can be used for different genomes like single[1]stranded RNA (ssRNA) single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) and PCR offers several advantages such as the capability to detect a single target in complex mixtures this is highly significant and, rapid and specific detection of multiple targets, and the potential to detect unculturable pathogens, such as viruses and some bacteria and phytoplasma which are not been culturable so far. So this is the strength of the PCR that apart from the known pathogens or culturable microorganisms it can also detect unculturable microorganisms or certain fungi or bacteria or phytoplasma along with viruses.

So Genome extraction of pathogens could be done either following manual techniques or using commercial kits specially designed to extract nucleic acids from different types of plant material. So DNA extraction from the plant material is not a problem nowadays as we have apart from manual techniques we have diagnostic kits or extraction kits they are commercially available for genome extractione. All molecular detection methods for detecting plant pathogens are basically based on accurate design of oligonucleotides and probes. Target sequences can be found in using the Gen Bank Nicleotide Sequence Search program that is NCBI and where most of the genomic data is stored and one can very easily find out the genomic resources from NCBI site. For searching of nucleotide bases normally a tool known as BLAST is used and the program that is BLAST and program is designed for analysis of nucleotides. Specific nucleotide regions are selected and primers for specific DNA or RNA targets can easily be designed using this blast tool. Primers are designed to pair with unique DNA regions from target organisms for DNA amplification and detection. The presence of amplification product confirms the presence of the organism in the tested sample. The amplified product then visualized through agarose gel electrophoresis using a stain known as ethidium bromide (EtBr). However nowadays less toxic and more sensitive stains like SYBR GREEN is used for detection method using under UV irradiation. Generally, PCR can be performed in two to three hours but advanced systems can deliver results in few minutes as well.

So, briefly PCR reaction mixture is prepared in a PCR tube, it composed of the template DNA, then the primers that complements the target the regions of the template DNA. Then along with free nucleotides, then Taq polymerase for amplification and then the mix buffer, all these are incorporated into the PCR tube at a specific proportion and that PCR is run and the PCR cycles include the first step that is denaturing that is separation of the two DNA strains at high temperature which is at around 95o C followed by temperatures that is set for annealing of PCR primers which is set at around 55 degree Celsius however it depends on the handling temperature of the primers. Then the next step is primer extension and the temperature here set is 72 degree Celsius and this cycle is then completed for next 32 to 35 cycles and after the limited number of cycles it is the PCR product is verified by running through in the agarose gel electrophoresis and the presence of a band that indicates the PCR positive results that confirms the presence of the particular pathogen in that particular genome that is extracted from the plant tissues.

Another method is DNA microarrays. So DNA microarrays are promising high throughput tools and can detect multiple pathogen at the same time. This is the major advantage of DNA microarray. So, microarray consists of a solid matrix usually a glass slide on which oligonucleotide probes or other DNA fragments are placed in very precise locations at high density. So this is a typical example of a DNA microarray chip where the in that microchip this is the DNA probes that are arranged at specific locations at very high density. 06:46 The target DNA sequence in a sample then hybridized to the probes and detect by fluorescence. So the target DNA sequence is then hybridized with the probe and then it is detected with a fluorescence light. The advantage of microarray based detection is the combined powerful nucleic acid amplification strategies with a massive screening capability resulting in high level of sensitivity, specificity and high throughput capacity. It can detect many different pathogens in a single assay so, that is why DNA microarray is also being used to detect multiple pathogens from a single species.

Another important DNA based technique is known as FISH in short in full it is known as Fluorescence In Situ Hybridization. This technique used 16S or 23S ribosomal( rDNA ) oligonucleotide probes labelled with fluorescent dye in combination with fluorescence microscopy. The FISH probes of size (20-30mers) recognized the pathogens in plant tissue cells, fixed in a microscopic slide and hybridized it with target gene in the pathogen in the plant samples. The probe target hybridization can visualized by fluorescent light. FISH was successfully used with probes to target the 23S rDNA to detect Ralstonia solanacearum in potato peels. So this is an another DNA based technology where a fluorescent probe is hybridized to a specific location on the microbial or pathogen genome and then it is visualized under fluorescent or confocal microscopy to see that whether that particular fluorescent probe is visible or not. Visible of fluorescent tag is confirmatory for the presence of that particular genome that is targeted. This could also be used to detect fungi and viruses and other endosymbiotic bacteria that infect plants. The high affinity and specificity of DNA probes provide high single cells sensitivity in fish because the probe will bind to each of the ribosomes in the sample. However the practical limit of detection lies in the range of around 103 CFU/mL. So this level or this limit should be achieved before observation under FISH or under fluorescent microscope. In addition to the detection of culturable microorganisms that cause plant diseases FISH could also be used to detect yet to be cultured so-called unculturable organisms in order to investigate complex microbial communities. So the probe can be designed to target even those un culturable microbes which are yet to be cultured. Further, FISH can also be used for detection of pathogens in which plants symptoms are yet to be produced. For example, here the downy mildew pathogen in impatiens plant was detected by using the fish technology. So these are the presence of sites of downy mildew in the impatiens leaves and this is again a opportunity to identify pathogen before its production of symptoms.

So, in short we have seen that nucleic acid-based technologies can be very successfully deployed in pathogen diagnosis and detection of even unculturable or yet to be cultured or even nonculturalable entities like viruses could be detected very successfully in presence even before production of the symptoms by the plants and so that it helps the farmers or growers to take adequate control measures at threat or appropriate time before the pathogen cause severe damage to the crop plants.

So, with this we have come to an end of the nucleic acid-based technologies and in the next topic we will be looking into the sort interfering like RNA based technologies.

Thank you very much