LAMP primer design

Nucleotide sequences in the four groups of CTX-M β-lactamase genes were selected from the National Center for Biotechnology information (NCBI). The accession numbers of these sequences were X92506.1, Y10278.1, AF255298.1, AY005110.1, AF305837.1, AY044436.1, AY080894.1, AF488377.1 for the CTX-M group 1; AB770487.1, Y14156, U95364, AJ005044, AJ005045.1, AJ416344.1 for the CTX-M group 2; AF189721.1 for the CTX-M group 8; AF174129, AF252623, AF252622, AY029068, AY033516, AJ416346.1, AY143430.1, AY156923.1 for the CTX-M group 9. These sequences were aligned using BioEdit 7.2.5 software [20]. Consensus sequences were analysed with the LAMP primers design software “Primer Explorer V4” software (http://primerexplorer.jp/e/) and specific primers were automatically designed. The four basic primers used for LAMP were a forward inner primer (FIP), a backward inner primer (BIP) and two outer primers (F3 and B3) [14]. One or two Loop primers (LF and LB) were designed for each target. These last are optional but can be used to accelerate the reaction time [21]. Primers were validated using BLAST software (http://www.ncbi.nlm.gov/BLAST) to ensure their specificity. The list of used primers is shown in Table 2.