5 Advanced DNA-Based Point-of-Care Diagnostic Methods

Transcript

Hello, welcome to the last talk of this week. That is DNA-based advanced point of care diagnostic techniques. So point of care techniques are gaining popularity because they don’t require sophisticated equipments. They are able to make immediate decisions and they are very easy to handle. So point of care diagnostic essays performed were a very cheaper rate as well and that is why they are also in very high demand. We know that PCR based techniques, although they have several advantages but certain limitations of PCR based technologies is that this technology cannot be taken directly into the fields and it requires electricity and other supporting devices. So it’s seriously limits its adequacy for point of care applications. As an alternative, isothermal DNA amplification methods are ideally suited to overcome this limitation of PCR. For instance, Isothermal amplification combined with lateral flow stripes and portable fluorometers have been successfully used for point of care, detection of pathogen DNA. so point of care, DNA extraction methods. In plant tissues DNA extraction method requires ability to efficiently remove in number of chemicals that can inhibit the DNA amplification reaction. So this is very important, very critical that to have a successful PCR run, we need to have a DNA extracted properly from the plant samples and remove all the chemicals that can inhibit its amplification. So lateral flow device in short, we call it as (LFD) DNA extraction method has been reported as rapid and efficient point of care testing and has been successfully used in plant pathogen detection. So this LFDs are being very useful in point of care detection of plant pathogens because it requires very less amount of substrate as well, less, very less time to get the DNA. This method involves sample disruption in extraction buffer using metal ball bearings before transferring the lysate into the release pad of a LFD nitrocellulose membrane.

The technology is very simple. Take the sample, put it into the sample buffer in the tube that contains two ball-bearings, that is metallic origin and then it crushes the tissues in a way and the tissue fluid is then transferred to the lateral flow device, then nitrocellulose membrane. Then small piece of membrane is excised and added into the DNA amplification reaction such as PCR or other isothermal amplification methods. So the membrane binds the DNA that is present in the tissue and this small portion of the membrane is still transferred to the PCR tubes or isothermal amplification tubes and this serves as a DNA that is present in the sample. The isolated DNA is very stable on the membrane at room temperature, which allows the extraction to be performed in the field condition. So that is why the point of care DNA extraction methods using nitrocellulose membrane has been so popular that it binds the DNA, it does not require any specific temperatures to be maintained and that’s how it is being used for point of care detection. Another, method is use of simple cellulose-based dipstick that allows plants sample processing as little as 30 seconds. Plant tissues are here basically macerated by shaking in a tube containing extraction buffer and 1 to 2 ball-bearings for 8to10 seconds. Then cellulose dipstick is inserted in the tube containing the sample before washing it three times in a second tube containing wash buffer, and finally into a tube containing the amplification mix. So this is again a very simple method, where does the cellulose based dipsticks are immersed, inserted into the plant tissues. And then it is macerated by the help of the extraction buffer and the ball[1]bearings. It is just done for 8 to 10 seconds and then the cellulose dipstick when inserted into the tube, then it adhered the sample and into it and then after washing it in a second tube then that dipstick can indirectly transferred to the PCR or amplification mixer.

So the technology works efficiently in multiple cultivated species including rice, wheat, tomato and sorghum as well as some difficult to isolate plant species like leaves from mature trees of mandarin, lime and lemon. So it can be used to detect pathogen DNA as well as RNA from infected tissues and works with multiple amplification methods such as PCR, LAMP and RPA. So this is another very easy point of care, DNA extraction method using cellulose based dipstick. So application of nucleic acid, isothermal amplification techniques in plant disease detection. So although PCR is highly sensitive and robust, it is constrained by a number of technical limitations. For instance, specificity is highly dependent on the primers used and its inherent sensitivity makes it prone to false positives due to sample cross contamination. So PCR, although it’s a very useful and sensitive kit which is able to detect pathogen, which is present in a very low amount, but it has certain limitations and besides PCR also requires electrically powered equipment to perform the thermal cycling, which limits its use of point of care diagnostics. A number of alternative isothermal techniques are now available that can obviate the need of a thermal cycler.

So one of those isothermal technique is Loop-mediated isothermal amplification, in short, it is known as LAMP. So it has widely been used to its high efficiencies, specificity, simplicity and quickness. LAMP requires two long outer primers and two short inner primers that recognize six specific sequences in the target. DNA. The first inner primer containing sense and antisense sequences in the DNA will hybridize the target sequence and initiate DNA synthesis. In the next step, the outer primer carries out the stranded-displacement DNA synthesis and produces a single stranded DNA which work as a template for the second inner and outer primers producing a DNA molecule with a loop structure. The unremitting cycling, reaction accumulates products with repeated sequences of target DNA of different sizes. The reactant tube is incubated at 63o C to 65o C in a regular laboratory water bath or heat block that helps in maintaining a constant temperature.

So this is one of the greatest advantages of this particular technique. We hear that we don’t require different temperature regimes as it is in the case of PCR where we require temperature for denaturation of DNA, then we required temperature for annealing of primer, and then we required a different temperature for extension of primer. But here we require only one temperature that is around 63 to 65 degrees Celsius, which can be maintained in a laboratory water bath or heat block. The amplified product can be detected by naked eyes as a white precipitate or a yellow green color solution after addition of SYBR green to the reaction tube. So here we don’t require even the gel electrophoresis or ETM bromide staining for detection of the PCR amplification product. Here the product can be visualized as white precipitate on the tube by even naked eyes or if we add SYBR green, then reaction mixer turns yellow, green in color. So this is a confirmation color change is a confirmation of the positivity of the reaction with the primers. There are three major advantages of this particular tactic. First, it can be carried out at a constant temperature with a short reaction time. This rapid isothermal process makes it ideal for point of care detection of plant pathogens in the field. Since it does not require any electrical support for running the reaction as well as we don’t require any gel electrophoresis system to visualize the outcome reaction. And that is why it is a very good tool for use of point of care, detection of plant pathogens. Secondly, it is very high amplification, efficiency and sensitivity as it generates large amount of PCR product with low amounts of input DNA.

And finally, this method is relatively cost effective. As it requires simple equipment to perform the assay. Furthermore, there have been reports that LAMP generates amplicons with several inverted repeats, which could be potentially used to increase the sensitivity in hybridization assays such as LAMP-ELISA hybridization and LAMP incorporated with colorimetric gold nanoparticle hybridization probes. So formation of the loops is also been considered to be a positive outcome of LAMP reactions because in techniques like LAMP-ELISA hybridization and LAMP incorporated with colorimetric gold nanoparticle hybridization process, it helps a lot. So in brief we can see the reaction procedure of the LAMP technique where this is the template DNA and this is the first forward internal primer that cleaves and in the complimentary site. And then it extends to this direction. There by forming this particular strand and this strand is now used by second primer that is backward internal primer. Then it extends to this directions and lead this synthesis of this new strength. And finally, since this section is complimentary to this, this will come and form a loop and this is complimentary to this region so this will come and form a loop. And this is the second loop it generates. And now this second primer for the second primer, this is exposed and then outer primer is then role comes into play. Then it starts amplifying from this point to the other point and it strands , the particulars in new strain. And then here the second outer primer binds and then it amplifies through this portion till this and it produces the new strength. So this is how the amplification process takes place in case of LAMP technique where two internal primers and two outer primers, they recognizes six sites first, second, then third, fourth, fifth, and sixth. So all these sites are recognized. And this LAMP technique is very useful for detection of plant pathogens under field conditions.

The integration of LAMP with electro chemical sensors offered a robust platform for pathogen detection as it was highly sensitive, detecting as low as 10 copies of the pathogen genomic DNA. So this is again, we can see that it is highly sensitive as only 10 copies of the pathogen, genomic DNA, sufficient for successful electrical chemical sensor of the LAMP process. So LAMP by sensor technology has a strong potential for infill testing, detection and identification of plant diseases. So we have seen here that various advanced DNA-based point of care diagnostic methods and one among them is the Loop mediated amplification of the DNA. And this technique is so popular nowadays as it requires very unsophisticated equipment, but the outcome of the process is very sensitive and reliable and it does not even require confirmation gel electrophoresis. And that is why this LAMP technology is now rapidly being used by plants disease diagnosticians to detect various plant pathogens and directly at field level. So with this, we have come to an end with a particular point of care technology that is based on DNA identification and we have come to end up the third week as well. In the fourth week we’ll be meeting again with the new topics on recent advances in diagnostic technologies. Till then, have a good time.

Thank you very much.