Let it be any disease, ranging from a fever (which is the symptom of many diseases) to HIV, diagnosis at an early stage is very important for giving specific treatment.Whenever I go to a doctor, he asks me things like, stomach pain, fever, body pain, head ache, running nose, throat pain etc., then, he checks pulse, then he uses his stethoscope, he then gives all the possible antibiotics! Yes, really, this happens in most cases. Then, if my problem is not fixing up in a week, he'll ask me to take a blood test, this, that, and, all!
Nothing wrong in this, because he can't ask everyone coming to him to take blood test at the very first sitting, then, no one will visit him back saying he sucks out blood all the time! But, it is okay to have a blood test after a week, but, not after a month! Diagnosing a disease after it had reached a severe stage is comparatively less significant than diagnosing at an early stage. So, how to diagnose?
Nothing wrong in this, because he can't ask everyone coming to him to take blood test at the very first sitting, then, no one will visit him back saying he sucks out blood all the time! But, it is okay to have a blood test after a week, but, not after a month! Diagnosing a disease after it had reached a severe stage is comparatively less significant than diagnosing at an early stage. So, how to diagnose?
Antigen - Antibody!
Antigen and Antibody interactions are mainly used for diagnosing most of the diseases. When you give your blood sample to your doctor, he gives it to his lab, there they check for the presence of particular antigen in your blood. For example, if you have typhoid, surface proteins of the causative organism (Salmonella typhi) will act as antigen and when antibody (specific protein produced by your body against the antigen) is added to the infected blood sample antibody - antigen interaction occurs, which could be detected by various techniques.Possible methods for diagnosing a disease!
There are several methods available for diagnosing a disease, but, most of them are based on the antigen-antibody interaction. Here I had listed a few methods of diagnosis, which I know:
1) ELISA - Enzyme Linked Immunosorbent Assay!
This method is based on the antigen antibody interaction. Two antibodies are used for detecting the presence of a single antigen. But, both the antibodies won't be directly reacting with the antigen, one antibody (primary) will be reacting with the antigen, and other antibody(secondary -which was raised against the primary antibody) will react further with the primary antibody used, producing color based on the enzyme linked with it. Generally done using 96 wells plate.
2) Western Blot!
This is also done for finding the presence of a specific protein (in diagnosis, the protein is antigen) in a given sample.This is done generally after doing SDS PAGE and transferring the protein separated in the gel to nitrocellulose membrane followed by similar technique of adding primary and secondary antibody, which will show color if there is infection.
3) PCR - Polymerase Chain Reaction
Though the presence of a particular gene could be detected using Southern blot, the most sensitive technique used for diagnosing a disease based on genes is PCR. Quantitative PCR could be done for even finding the severity of the disease based on the concentration of a particular gene of the causative organism.
I prefer to go with PCR than any other technique, but, when there is no thermo cycler in your laboratory it's better to go with ELISA! There are also other techniques like latex agglutination and microscopic observation of samples, but, the best way is PCR!
What do you say? PCR?
I appreciate your efforts and enthusiasm for these posts. It is good that you have an overall idea of these techniques. But I would recommend you reading more research papers. On any day, if you know the protein of pathogenesis or response that you are looking for then Western blot is gold standard.
ReplyDeleteFor example: Proteins from the HIV envelope gp41, and gp120/gp160.
Proteins from the core of the virus: p17, p24, p55
Enzymes that HIV uses in the process of infection: p31, p51, p66
But possibly could not be employed in a hospital setting as it needs skilled labor and it is time consuming.
Moreover just PCR is not sufficient to conclude any infection. The genes of immune response or infection are broad spectrum and are not unique. Hence usually a qRT PCR is important if the gene is unique for a disease. In case the treatment regime needs to know the species of the infecting microbe then PCR is not at all a standard one. In such cases, if doubted for Fungus or any eukaryotic infection, then a PCR using 18s rRNA primers followed by sequencing to find the sequences to get an analysis by BLAST (blast.ncbi.nlm.nih.gov) to find the phylogeny will be ideal to know if it is Fusarium or Aspergillus to start a prompt treatment. In case of prokaryote, 16s rRNA to find the organism would be ideal. So just a PCR won’t do.. “I prefer to go with PCR than any other technique; the best way is PCR” - these statements are very layman like and must be reconsidered and the author is suggested to discuss with experts and read more research articles. Interest is saluted!
Your reply or disagreement or inputs welcome and will be appreciated..
Real Time PCR could be used for even finding out the different subtypes which are causing the infection, right? Upto my knowledge, specific genes of different subtypes of disease causing oganisms are being found and when we target those genes and do PCR, it is easy to confirm and diagnose, right? I believe PCR is a very powerful tool in diagnosis.
Delete// if doubted for Fungus or any eukaryotic infection, then a PCR using 18s rRNA primers followed by sequencing to find the sequences to get an analysis by BLAST (blast.ncbi.nlm.nih.gov) to find the phylogeny will be ideal to know if it is Fusarium or Aspergillus to start a prompt treatment//
As you said here, PCR is involved in all the process of diagnosis and conformation of the infectious organism, I didn't try to give a very detailed note in this post, but an overview of diagnosis techniques available.
Thanks for spending your valuable time in commenting here. And, I'm still learning things and there might be mistakes (as I'm writing what I understood as mentioned in the disclaimer) in my articles. But, nothing won't be completely wrong, I believe.
Thanks once again :)
Thanks for your time and interest to respond here. Sorry for the delay in my response, I didn't notice your reply. Yes you are right, in certain cases sub types can be differentiated using qRT-PCR.
ReplyDelete//PCR is involved in all the process of diagnosis and conformation of the infectious organism//
What I said in the first comment was "just PCR" alone would not be sufficient as a powerful diagnosis tool. Moreover, if you ask for gold standard diagnostic assays then Western or ELISA would top.
I am sorry if I had sounded rude (did I?). As a budding youngster I see more of me in you through your writing. During my undergrad and post grad studies, I used to take things if it sounds reasonable to me. I don't want you to do the same mistake that I did! All I am suggesting is that you give it a more thought and try to justify if that particular thing is indeed the best choice.
I am learning things as well and I learn from some of your writings here too. I never said that what you wrote is wrong. All I wanted and want to say is put facts to convince statements. May be make references in brackets as much as possible. In science you need facts and evidences to prove than belief..
Keep writing. All the Best :-)
:) No, you are not rude :D Did I sound rude? :O It's my pleasure that you are commenting here :) Thank you so much, Will follow what you said from next time :)
DeleteThank you once again!