Discussion created on 03/23/2012

Loop-mediated isothermal amplification of poultry

Forum: Loop-mediated isothermal amplification (LAMP) a breakthrough in diagnosis of poultry pathogens?

Dear friends,

Viewing your reports regarding the occurrence of IBD, Mycoplasma, Marek's disease, IBH, ND and other important factors for poultry industry I would like to recommend the new and simple loop-mediated isothermal amplification method for the rapid and simple detection of many poultry pathogens.

I successfully applied this tool for diagnosis of Marek's disease and differentiation between different strains used in prophylaxis of MD. Similarly I used LAMP for the detection of waterfowl pathogens as goose circo-, and parvovirus. This method does not need to apply any sophisticated stuff and as I predict this replace well known PCR. Let me know if you ever heard about it in any applications. I am curious what is your opinion regarding the new alternative for molecular biology methods.
Cordially,

Greg

Grzegorz Wozniakowski
Doctor of Veterinary Medicine
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March 28, 2012
How LAMP is applied,in lab or at farm.Send details of LAMP.How efficient & accurate is this test.
Regards,
Dr Khuram
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Sreenivasa Bharath Sreenivasa Bharath
Doctor of Veterinary Medicine
March 28, 2012
Can i have the procedure of the test and it's cost implications
thanks
Dr.Bharath
Reply
March 28, 2012
Thank you for your interest. LAMP can by used even at farm. I haven't tested LAMP directly with samples of tissues or blood but some people succeed to do it. You don't need thermocyclers but only a water bath or dry block. If you are interested in the application of LAMP you may read my publication in Avian Diseases 55:462–467, 2011 entitled: Rapid Detection of Marek’s Disease Virus in Feather Follicles by Loop-Mediated Amplification". I also used this technique for the detection of goose parvo- and circovirus. An example of LAMP procedure is as follows:

“The LAMP reaction was carried out in 25 µL containing: 1× Pol buffer (50 mM Tris- HCl pH 9.0, 50 mM NaCl, 5 mM MgCl2) , 1.6 M betaine, 1.5 mM of each dNTPs , 50 pmol each of inner primers FIP and BIP, 10 pmol each of outer primers F3 and B3, 25 pmol of F2 and F3 primer, 8U Bst DNA polymerase , 2 µL of DNA template (~50 ng) and deionized water. Tubes were then incubated in a water bath (65 – 68°C) for times ranging 45 to 120 min. The mixtures were then heated to 80°C for 2 min. After this step 1 x concentrated SYBR Green (Invitrogen) was added to the each sample. Samples were observed using a UV transilluminator to observe color change during LAMP. Tubes were photographed with Canon camera.”
Please let me now if you need further explanations. The specific procedure should be designed for the particular pathogen. However this method is quite flexible so the protocol may be universal as well.

Regards,

Greg
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March 28, 2012
I forgot about cost implications. The estimated cost of 1 sel-made test is below 2 USD. Since you don't need thermocycler it is a low-cost method. The commercial tests are accessible but they are more expensive (about 13 USA/sample).

Regards,

Greg
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Sreenivasa Bharath Sreenivasa Bharath
Doctor of Veterinary Medicine
March 28, 2012
thanks for the details, do you have the commercial test kit suppliers address? if so,plz can i have it.

i will also go through your article
thanks again
Reply
March 28, 2012
For example you can find the kits called Looplamp here http://www.mastgrp.com/catalogue_products_fulldetails.asp?SubProduct_Type=12046&cat=1&product=LMP205.

Another company called Eiken produce kits for LAMP but I encourage you to use self-validated method using any Bst or Bsm polymerase.

Best regards,

Greg
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Cahit Ayata Cahit Ayata
Veterinary Doctor
March 29, 2012
i have found this topic very interesting, i will read your publication right now.
Reply
Dr Muhammad Arshad  Manj Dr Muhammad Arshad Manj
Doctor of Veterinary Medicine
March 30, 2012
mr Greg in pakistan from where it can obtained. plz elaborate in detail the lamp method
Reply
Nadir Alloui Nadir Alloui
Veterinary Doctor
April 3, 2012

Dr Greg ; Thank you very much for the information. Is it possible for me to come and do a short internship this method of diagnosis in Instytutcie Veterinary in Pulawy.

Reply
April 3, 2012
I regret but the technique is quite fresh and we haven't introduced it as routine laboratory procedure. As far as I am concerned I know that Polish company called Novazym offers great LAMP kits ready to use. See here http://www.novazym.ehost.pl/esklep4/isothermal-mastermix-fluorescent-dye-400-reakcji-p-173.html
They also design LAMP primers. I think in the nearest future I am going to organize LAMP workshops in Pulawy, Poland if you are interested I will let you know. As I found there is another "better" option for Bst polymerase used in LAMP. It is called SSD polymerase. It facilitate to give results in 15 minutes!
I have tried it and indeed that's true.

All the best,


Greg
Reply
Cahit Ayata Cahit Ayata
Veterinary Doctor
April 4, 2012
We are currently doing tests for ART (TRT), MG, IBD, IB and ND by using elisa kits. Due to elisa prodecure there is a risk for false pozitive and takes lots of time if we need to re-do the test. Do we have a chance to test for those same pathogens by using LAMP kits? If we do, what kind of samples do we need to collect and how is the test procedure for them?
Reply
April 4, 2012
As I remember LAMP tests have been successfully developed for diagnosis of ND, IBD, IB, MD. You can access the publications about them below. So far LAMP has been not developed for TRT. So it is also a new challenge to find a new method.

http://vdi.sagepub.com/content/21/6/841.long
http://www.sciencedirect.com/science/article/pii/S0890850809000644
http://www.sciencedirect.com/science/article/pii/S0890850809000644
http://www.ncbi.nlm.nih.gov/pubmed/22017048

Hope these data is helpful for you. In previous posts you can find links to reagents for LAMP.

Greg
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