DNA

Part:BBa_K3286230

Designed by: Niels Appelman   Group: iGEM19_Wageningen_UR   (2019-10-19)
Revision as of 08:18, 27 September 2022 by Zxy0331 (Talk | contribs)


LAMP detection site for Xylella fastidiosa (part of RimM gene)

This DNA sequence is recognized by the LAMP primers used most commonly for detection of Xylella fastidiosa, as published in the paper "Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications." by Harper et al. 2010. If one transforms it into a bacterium, this bacterium can be used as a model organism for the detection of X. fastidiosa by LAMP.

It consists of bases 72-444 of the RimM gene as published on: https://www.genome.jp/dbget-bin/www_bget?xfa:XF_0108

contribution

Loop-mediated isothermal amplification method (LAMP) is a new isothermal nucleic acid amplification method invented by Notomi et al. Using a DNA polymerase with strand displacement activity (Bst DNA polymerase) under isothermal conditions (60-65 °C), the nucleic acid amplification reaction can be completed within 1 h, and the target fragment can be amplified 109 times. The method has the advantages of strong specificity, high sensitivity, high amplification efficiency, time saving and simple operation, and has obvious advantages in nucleic acid detection. At present, the product detection methods of ring-mediated isothermal detection technology mainly include turbidity method, dye method, electrophoresis method and nucleic acid test strip, but these methods are prone to cause cross-contamination.

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None