Difference between revisions of "Part:BBa K3286230"

Latest revision as of 06:09, 30 September 2022

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 by 2022 iGEM Team YkPaO

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.

3. Place the microtube into a 65C water bath and heat it for 30-60 minutes.

4. Place the tube in a gradient thermo cycler and heat at 80C for 10 minutes to denature all DNA polymerase

Amplifying oligo DNA sequences by LAMP

Figure 1. 1.5% TAE agarose gel electrophoresis to verify the amplified oligo DNA fragments.

The lamp is a novel nucleic acid isothermal amplification method, which is widely used in the rapid detection of bacteria, pathogenic microorganisms, parasites, and viruses. Compared with the currently widely used real-time qPCR technology, it requires four to six template-specific primers and DNA Polymerase with strand displacement activity, and the amplification is completed within 1 h under isothermal conditions; The corresponding detection dye can determine whether there is an amplification product. In order to make our detection kit more suitable for home portable use, we tested amplified oligo DNA with the lamp method, which is used as an alternative to the PCR amplification method. The experimental results show that there is our target band at the position of 500bp, indicating that the scheme of amplifying oligo DNA by the lamp method is feasible. This provides strong experimental data support for our development of portable detection kits.

Sequence and Features