Primers and molecular beacon probes

The LAMP primers (F3: outer forward primer; B3: outer backward primer; FIP: forward inner primer; BIP: backward inner primer) were designed in Primer Explorer V4 (http:/primerexplorer.jp/lamp). Two additional loop primers (LF: loop F, LB: loop B) were designed to accelerate amplification. The fluorescent nucleic-acid probes in MB-LAMP (LBP and LFP) were directly modified from LB and LF (Fig. 8). As shown in Fig. 8, the fluorescent nucleic-acid probe LBP was modified from the LB; the last 19 nucleotides in the LBP sequence are identical to the LB and form the heads as well as one arm of the molecular beacon. The first six sequences of the LBP are the reverse complement of the last six sequences and form the other MB arm. The fluorophore and quencher are linked to each MB arm, respectively. The LFP was transformed from the LF in the same way. We chose the pair of LBP and LF in our MB-LAMP experiment randomly. Table 3 shows the LAMP primers and molecular beacon probes used in this manuscript. Table 1 shows the long and short molecular beacon probe sequences used in the experiment for MB length optimization (Fig. 2). One of the arms and the head of LBP-1 were the extended sequences of LB, which was part of the ompW gene. We only needed to use the 29-base sequence from the LB and to mark the last six bases and their reverse complementary sequences as arms. Finally, we connected the fluorescence and quenching group. Similarity, one of the arms and the head of LBP-2 is a part of LB. We used the first 9-base sequence from the LB and marked the last six bases and their reverse complementary sequences as arms. After connection of the fluorescence and quenching groups, LBP-2 was designed well.

The fluorescent nucleic-acid probe LFP LBP were modified from the LF LB as described in text, respectively.