3 Microneedle Based Diagnosis

Transcript

Hello!! welcome to the third talk of this week and that is Microneedle based diagnosis. We have already discussed about nucleic acid-based technologies which we know that it is very specific and very important but in field diagnosis nucleic acid-based technologies has challenges and to overcome these challenges there is constant improvement of this particular technology where it is being simplified and modified and one among this technology is use of micro needle for DNA extraction for direction of plant pathogens from infected plants under field condition. So in field molecular diagnosis of plant diseases via nucleic acid amplification (NAA) is currently limited by cumbersome protocols for extracting and isolating pathogen DNA from plant samples. To address this challenge a rapid plant extraction method has been developed using a disposable polymeric Microneedle (MN) patch. By applying microneedle patch on plant leaves amplification assay-ready DNA can be extracted within a minute from different plant species. Microneedle extracted DNA has been used for direct polymerase chain reaction amplification of plant plastic DNA without purification. Further, more using this patch device extraction of plant pathogen DNA for example from Phytophthora infestans from both laboratory inoculated and field-infected leaf samples were performed for detection of late blight diseases in tomato.

So microneedle extraction achieved hundred-percent detection rate of late blight infections for samples after 3 days of inoculation when compared to the conventional Cetyl trimethyl ammonium bromide that is (CTAB) method based DNA extraction method as gold standard and 100% rejection rate for all blind field samples tested. So the microneeddle based extraction method is comparable with most common and most preferred DNA extraction method that is CTAB method and that is why microneedle based DNA extraction method is having tremendous potentiality to be used under field conditions in coming years. This simple cell lysis[1]free and purification-free DNA extraction method could be transformative approach to facilitate rapid sample preparation for molecular diagnosis of various plant diseases directly in the field. The microneedle patch is postage stamp-sized it can directly be placed in a leaf and pressed if we see the microneedle patch then it comprises of small cone like projections which is basically very inexpensive polymers and this postage size stamp of microneedle patch is very convenient to use and this cone like structures of polymers are hardly .8 millimeters long. So when this patch is fixed or pressed against a leaf it then penetrates the leaf epidermis and the DNA is get adhered to the polymer from the healthy plant tissues of Mesophyll cells as well as infected tissues of Mesophyll cells comprising of pathogen DNA as well. So the DNA gets bind with the polymer of the microneedle patch and this patch can now be taken for PCR amplification. That microneedle method has advantages over CTAB method because DNA extraction by CTAB method requires a lot of equipment and it takes several hours. Further, CTAB extraction is a multi-step process involving tissue grinding to organic solvents and centrifuges. By contrast the new micro needle based DNA extraction technique involves only few steps and require only a microneedle patch and an aqueous buffer solution and it can extract DNA within few seconds or minutes. So this is a brief outline of the CTAB method which involves several processes and that is why it is very time-consuming and usually it takes three to four hours for extraction of DNA.

In comparison to the CTAB method microneedle patch can extract DNA within a minute. So for a farmer or a researcher can directly apply the microneedle patch to the plant they suspect to be diseased, then hold the patch in place for a few seconds and then peel it off. The patch is then rinsed with the buffer solution washing genetic material of the micro needles and into a sterile container. The entire process takes just about a minute. The micro needle techniques purity levels were comparable to other validated laboratory methods of DNA extraction, we have already talked about it and the most importantly the slight difference in purity levels between the micro needle and CTAB samples did not interfere with the ability to accurately test the samples by a PCR or LAMP assay. So although the quality of DNA may be compromised to some extent but it is not having any effect on PCR amplification or LAMP assay process and that is why microneedle based DNA extraction method is very much recommended nowadays for extraction of DNA directly from field Samples. Since DNA extraction has been considered as a significant hurdle to the development of on-site testing tools, the micro needle based technology has now given a solution to the problem. It is an integrated low cost field portable device that can perform every step of the process from taking sample to identifying the pathogen and reporting the results of an assay.

Further, Lateral Flow Device (LFD) could be used to accurately detect the pathogen DNA extracted through the micro needle technology. So both micro needle technology along with the little flow device can be used under field conditions so the problem regarding extraction of DNA is solved through micro needle based technology and the PCR based technology is replaced with little flow devices and that is how in-field the pathogen can be directly detected by using these novelapplications like micro needle based and patch as well as Lateral Flow Devices (LFD). So, we have seen the utility of microneedle based technology for extraction of plant DNA from the field samples and it is highly comparable to the gold standard CTAB method and it gives nearly hundred percent results from the 0extracted DNA that was used through microneedle patch. So with this we have 0 seen another novel technology of DNA extraction and this particular technologies need to be further popularized and need to be used in field conditions for saving time and resources and for quick identification of plant pathogens that is causing certain diseases under field conditions. so thank you very much in the next talk we’ll be talking about artificial intelligence in disease diagnosis.

Thanks.