- Home
- Protocols
-
Design of LAMP Primers
Request a Protocol
Ask a
question
Universal LAMP primers were designed for detection of A. baumannii, A. pittii, and A. nosocomialis using nucleotide sequences of the ITS region between 16S and 23S rRNA genes of respective strains. A total of 10 sequences (four sequences each from A. baumannii and A. pittii and two sequences from A. nosocomialis) were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/) using respective GeneBank accession numbers: AY601823.2, AY601826.1, AY601825.1, AY601824.1 (A. baumannii), EU030650.1, EU030647.1, AY601827.2, AY601829.1 (A. pittii), and AY601830.2, FJ360743.1 (A. nosocomialis). Using the BioEdit Sequence Alignment Editor (Informer Technologies, Inc.), a common sequence was aligned from the conserved location of these 10 sequences (Figure 2A). However, within this common sequence, different nucleotide bases were present in A. baumannii, A. nosocomialis, and A. pittii at position numbers 153 (G,G,A), 178 (T,C,C), 279 (T,T,G), and 308 (A,G,G). Nucleotide bases located at positions 153, 178, 279, and 308 were replaced by mixed bases R, Y, K, and R, respectively, and a consensus sequence was generated (Figure 3).
• Alignment of the above 10 sequences demonstrates that our consensus sequence is conserved across the diverse sequence of three species (Figure 2A). Hence, the consensus ITS 16S–23S rRNA sequence shown in Figure 3 was finally used to design the LAMP primers that included forward and backward outer primers (F3 and B3), forward and backward inner primers [FIP(F1c + F2) and BIP(B1c + B2)], and forward and backward loop primers (LF and LB). This consensus sequence was also aligned with the sequence of Acinetobacter calcoaceticus and non-ACB complex species (A. lwoffii, A. radioresistens, A. haemolyticus, and A. junii) to study the specificity of primers (Figure 2B).
• A total of 30 sets of outer and inner LAMP primers were designed using Primer Explorer V5 version software (Eiken Chemical Co., Ltd., Tokyo, Japan). The selection of primers was based on various criteria and included length (18–25 bp), melting temperature (Tm) (60–63°C for F1c and B1c and 55–58°C for F2, B2, F3, and B3), GC content (30–65%), and the distance between primers (120–180 bp for F2 and B2, 45–60 bp for F2 and F1c, 2–20 bp for F3 and F2, and 5–100 bp for F1c and B1c). Five sets of ITS primers were selected based on the stability of either end of each primer (using the dG value ≤−4.0 K cal/mol). However, positions of bases were shifted in order to design the loop primers manually and to fulfill the selection criteria. Finally, all the primer sequences were tested against the Basic Local Alignment Search Tool-National Center for Biotechnology Information (BLAST-NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure specificity.
(A) shows the alignment of sequence of diverse isolates of A. baumannii (N=4), A. pittii (N = 4), and A. nosocomialis (N=2) and demonstrates that the sequences are conserved across diverse isolates of three species except few locations. (B) shows the alignment of our consensus ITS 16S–23S rRNA sequence with the sequence of other Acinetobacter species (A. calcoaceticus, A. lwoffii, A. radioresistens, A. haemolyticus, and A. junii). The presence of the non-conserved region at locations from 140 to 210 (where our two to three primers are aligned) demonstrates that our LAMP primers were not specific for other Acinetobacter species.
A universal consensus sequence of the ITS 16S–23S rRNA gene that was used to design the LAMP primers for detection of significant members of the Acinetobacter calcoaceticus–baumannii (ACB) complex. Red color bases signify the mixed bases used in place of different bases present in A. baumannii, A. nosocomialis, and A. pittii. The sequences sites for ITS Ab1 and Ab2 LAMP primers are designated by upper and lower horizontal arrows, respectively. Right and left arrows indicate sense and reverse complementary sequences that were used.
The bla OXA-23-mediated carbapenem resistance was detected using OXA-23 LAMP primers described previously by Yamamoto et al. (2015).
Performance of the LAMP assay was validated with PCR. Forward and backward outer LAMP primers (F3 and B3) of ITS and OXA-23 were used for the PCR assay. The primers used in this study were procured from Integrated DNA Technologies, Inc., United States, in lyophilized form (Table 1). Primers were reconstituted using nuclease-free water and stored at −20°C.
Primers used in the study.
F3, forward outer primer; B3, backward outer primer; FIP, forward inner primer; BIP, backward inner primer; LF, loop forward primer; LB, loop backward primer. MP primers are used for amplification of ITS 16S–23S rRNA for identification of clinically significant members of the Acinetobacter calcoaceticus–baumannii (ACB) complex and blaOXA-23 gene for detection of carbapenem resistance.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Post a Question
