Hello Shazleen, 

Ok, it is maybe worth to do 72-74 degree, it might help 5-10 min delay of non-specific amplification. You can pre-add hydroxy naphtol blue to visualize the amplification in the reaction buffer with final conc. of 120uM, you will see the pale blue in solution upon amplification, so that you don't have to open the tube to monitor the amplification. If you are working with only 4 primers, 45 min seems to be good number.

for the reaction settings, it is worth separating the negative control tube-strip and positive control tube-strip, and prepare the negative control mix first. Do the assay n=4-8 per one strip. If you have inconsistent results on negative control, it means that the primer is non-specifically binding to the wrong place, so consider designing another primer set. If you have the amplification on all tubes, think about the contamination (during handling, or in template DNA (maybe during DNA extraction).

To avoid primer dimer artifacts, it might be better keeping reaction mix tube always on 4 degree.

You raise the reaction temperature, separate the tube-strip for negative control and the unknown, keep reaction mix on ice, and still have problems on negative control, finally it comes to the reaction mixture modification, and design/quality of primers.

for the reaction mixture, I use almost the same conc. of DNA primers, dNTP as written in the manual of OmniAmp DNA pol. For betaine, Bst DNA pol can accept more betaine so the conc. is 1.6 M. For the buffer modification, first thing that I would modify is betaine concentration, it works, but in such case the optimal Tm will change so re-optimize the reaction temperature. For the primers, I use FIP and BIP purified with cartridge and F3, B3, just desalted.

Sorry for the long post, hope that it can be any help for you.

Hope this is useful for you to help you designing primers for LAMP

http://loopamp.eiken.co.jp/e/lamp/primer.html

It is not solely on your primer design. It is a fundamental prblem with most LAMP assays. Check out this article:

Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loopmediated isothermal amplification (UDG-LAMP)

Chem. Commun., 2014,50, 3747

There is always possible to get contamination. However, as long as your negative control and positive control revealed correct results you are fine. As suggested by George Kyei-Poku it is ideal to use UDG to get read of carryover contamination.

 Dear shazleen mohtar,

i m MSc student of medical parasitology and i use LAMP in my thesis....maybe from primers but  you know that one of the problems in LAMP is high contamination ِof DNA in work place that lead to false positive so  i recommend , you change former work place and go to place that be Free from contamination of dna ,even change your White lab coat , then you do LAMP again

best wishes

Could you tell me what the reaction temperature and the reaction time are. I think that sometime you are catching the primer dimer or those artifacts. Sometime you can raise the reaction temperature and solve the problems, but it depends on the enzyme that you use.

I already change the working area. We are just moving into a new lab in a new building. However, I still got the false positive result? Maybe I need to optimize the reaction mixture.

Dear Yoda,

I am using OmniAmp DNA polymerase, my reaction temp is 70 with incubation time 45 minutes. 

Hello Shazleen, 

Ok, it is maybe worth to do 72-74 degree, it might help 5-10 min delay of non-specific amplification. You can pre-add hydroxy naphtol blue to visualize the amplification in the reaction buffer with final conc. of 120uM, you will see the pale blue in solution upon amplification, so that you don't have to open the tube to monitor the amplification. If you are working with only 4 primers, 45 min seems to be good number.

for the reaction settings, it is worth separating the negative control tube-strip and positive control tube-strip, and prepare the negative control mix first. Do the assay n=4-8 per one strip. If you have inconsistent results on negative control, it means that the primer is non-specifically binding to the wrong place, so consider designing another primer set. If you have the amplification on all tubes, think about the contamination (during handling, or in template DNA (maybe during DNA extraction).

To avoid primer dimer artifacts, it might be better keeping reaction mix tube always on 4 degree.

You raise the reaction temperature, separate the tube-strip for negative control and the unknown, keep reaction mix on ice, and still have problems on negative control, finally it comes to the reaction mixture modification, and design/quality of primers.

for the reaction mixture, I use almost the same conc. of DNA primers, dNTP as written in the manual of OmniAmp DNA pol. For betaine, Bst DNA pol can accept more betaine so the conc. is 1.6 M. For the buffer modification, first thing that I would modify is betaine concentration, it works, but in such case the optimal Tm will change so re-optimize the reaction temperature. For the primers, I use FIP and BIP purified with cartridge and F3, B3, just desalted.

Sorry for the long post, hope that it can be any help for you.

Yes, this is do to carryover contamination of amplified LAMB product, because LAMB assay produce large copy of product than PCR. You will get contamination while gel running or opening of tubes after reaction. Avoid gel running of LAMP product this will give more contamination. So, please change all reagent for LAMP, including enzyme (stock) and primer (stock) and do again. Please do not open tubes after reaction, lot of methods are published to do LAMB with opening the tube. Better use filter tips and change your pipette.