Apr 15, 2020
Detection of five bacterial pathogens of rice by multiplex PCR
- 1IRD, Montpellier, France
External link: https://doi.org/10.1371/journal.pone.0232115
Protocol Citation: Martine Bangratz, Charlotte Tollenaere 2020. Detection of five bacterial pathogens of rice by multiplex PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bcpaivie
Manuscript citation:
M. Bangratz, I. Wonni, K. Kini, M. Sondo, C. Brugidou, G. Béna, F. Gnacko, M. Barro, R. Koebnik, D. Silué, C. Tollenaere (2020) Design of a new multiplex PCR assay for rice pathogenic bacteria detection and its application to infer disease incidence and detect co-infection in rice fields in Burkina Faso. PLoS One.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2020
Last Modified: April 15, 2020
Protocol Integer ID: 33218
Abstract
This multiplex PCR protocol is used for the detection of five bacterial pathogens of rice : Pseudomonas fuscovaginae, Burkholderia glumae and gladioli, Pantoea spp, Xanthomonas oryzae, Sphingomonas spp.
Materials
MATERIALS
AgaroseMerck MilliporeSigma (Sigma-Aldrich)
5X Hot Firepol Multiplex Mix Ready to load
(NH4)2SO4 160 mM
TBE buffer
ethidium bromide
- Applied Biosystems Veriti 96-Well Thermal Cycler
- Oligonucleotides :
Pseudomonas fuscovaginae (amplicon size: 710 pb)
Pfs207-F : CAGTTCGATGGTCTGGGAAT
Pfs207-R : GGGACTGGTAAAGCACGGTA
Burkholderia glumae and B.gladioli (amplicon size: 508 pb)
toxB_F : GCATTTGAAACCGAGATGGT
toxB_Rd : TCGCATGCAGATAACCRAAG
Sphingomonas spp.(amplicon size: 435 pb)
Sphingo_KK_F1: CGGCTGCTAATACCGGATGAT
Sphingo_KK_R1: AGGCAGTTCTGGAGTTGAGC
Xanthomonas oryzae (amplicon size: 331 pb)
Xo3756F : CATCGTTAGGACTGCCAGAAG
Xo3756R : GTGAGAACCACCGCCATCT
Pantoea spp (amplicon size: 263 pb)
PAN_KK263F : GCGAGCCAATCGACATTA
PAN_KK263R : CGAGTAACCTGAGTGTTCAG
Before start
- Wear clean gloves
- Clean and disinfect the PCR cabinet
- Defreeze DNA samples and reagents
- Gently mix the DNA samples and the 5X Hot Firepol master mix
- Vortex the (NH4)2SO4 and the oligonucleotides
- Spin down the DNA sample and all reagents and keep them on ice
- Mark the 1.5 ml "Mix" tube and the 0.2 ml tubes for the PCR . Keep them on ice
The amounts described in this protocol are for one sample. Calculate the quantity you need based on your number of sample. Always prepare 10 % more mix.
Prepare a 2% agarose gel. Load 10µl PCR product and perform the electrophoresis at 100 Volts for 90min
Observe the gel under UV light after a ethidium bromide bath