Primer sequence:

16S:

F: CCGTAAGGAGGAGGAAGGT
R: GCAACATGGCTGATTTGCG
FIP: TTGCTTCTCTTTTGTGCACCCCCAGTCAAGTCATCATGGCCCTT
RIP: ACTGCGAAGTGGGGAGCCAATCTTTGCTTCATGCAGGCAGTT

cagA:

F: GATTTAATCAACAAAGACGCTC
R: TCTGCTTTTTCTTTGTCATCAGG
FIP: CCAACTTGTGAAAATTCGGTAACGGTAGAATCTTCCACAAAGAGCT
RIP: GTGTCCCATCAAAACGATCCGTAGGGGGTTGTATGgTATgTTCC

invA:

F:GGCGATATTGGTGTTTATGGGG
R: AACGATAAACTGGACCACGG
FIP: GACGACTGGTACTGATCGATAGTTTTTCAACGTTTCCTGCGG
BIP: CCGGTGAAATTATCGCCACACAAAACCCACCGCCAGG

ipaH:

F:GCTTCGACAGCAGTCTTT
R: GGCCAGGTAGACTTCTATCT
FIP: CCTTCTGATGCCTGATGGACCCACTGAGAGCTGTGAGGA
BIP: ATAATGATACCGGCGCTCTGCCCTCCAGAATTTCGAGGC


Procedure:

  1. Unfreeze 2× Lamp Master mix and primer on ice and mix the two reagents thoroughly.
  2. Create the reaction system by adding the following reagents in their assigned portions into a microtube.
    Reagent 25μl Reaction system Final concentration
    2×Lamp Master Mix 12.5 μl
    FIP (10μM) 2μl 0.8μM
    BIP (10μM) 2μl 0.8μM
    F3 (10μM) 0.5μl 0.2μM
    B3 (10μM) 0.5μl 0.2μM
    Template DNA 1.5μl 1-4 ng/μl
    DNA polymerase 0.5μl 0.16 U/μl
    Sterilized ddH2O 5.5μl -
  3. Place the microtube into a 65℃ water bath and heat it for 30-60 minutes.
  4. Place the tube in a gradient thermo cycler and heat at 80℃ for 10 minutes to denature all DNA polymerase.
  5. Test resulting DNA using gel electrophoresis:
    1. Prepare 1% gel electrophoresis by preparing the gel
    2. Pipette 3-5μl of LAMP resultant using a pipette and run gel electrophoresis at 180V for 10 minutes.
    3. Agarose electrophoresis was used for detection. Take 3-5μl amplification products, prepare 1% agar gel for electrophoresis detection, and lamp characteristic maps composed of different size bands can be seen.