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Rapid detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring bla(VIM-2), bla(IMP-1) and bla(OXA-23) genes by using loop-mediated isothermal amplification methods.

Kim HJ, Kim HS, Lee JM, Yoon SS, Yong D - Ann Lab Med (2016)

Bottom Line: The optimal reaction temperature and incubation time were determined by using a gradient thermocycler.Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2).These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance.

Methods: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO₄) by testing different concentrations of MgSO₄. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates.

Results: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles.

Conclusions: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.

No MeSH data available.


Related in: MedlinePlus

Determination of the detection limit of the loop-mediated isothermal amplification (LAMP) assays and the minimum reaction time. (A-C) Various amounts of template DNA (1 fg, 10 fg, 100 fg, 1 pg, 10 pg, 100 pg, 1 ng, or 10 ng) were used in the LAMP reactions. The amplification was performed with 2mM MgSO4 at 62℃ for 1 hr. (D-F) LAMP reactions were performed for a range of reaction times. After the reaction time indicated on top of the gels, the LAMP reaction was terminated by inactivating Bst DNA polymerase by incubation at 95℃ for 3 min. The reaction products were loaded onto a 1.5% agarose gel for analysis.
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Figure 3: Determination of the detection limit of the loop-mediated isothermal amplification (LAMP) assays and the minimum reaction time. (A-C) Various amounts of template DNA (1 fg, 10 fg, 100 fg, 1 pg, 10 pg, 100 pg, 1 ng, or 10 ng) were used in the LAMP reactions. The amplification was performed with 2mM MgSO4 at 62℃ for 1 hr. (D-F) LAMP reactions were performed for a range of reaction times. After the reaction time indicated on top of the gels, the LAMP reaction was terminated by inactivating Bst DNA polymerase by incubation at 95℃ for 3 min. The reaction products were loaded onto a 1.5% agarose gel for analysis.

Mentions: To assess the limit of detection (LOD) for each LAMP reaction, LAMP assays were performed by using a series of DNA template samples prepared from 10-fold serial dilutions of a stock with an initial concentration of 10 ng/µL. A typical ladder-like pattern was detected by using as little as 1 pg of target DNA for blaVIM-2 and 10 pg of target DNA for blaIMP-1 and blaOXA-23 (Fig. 3A-C). We then compared the LOD for each target gene in conventional PCR.


Rapid detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring bla(VIM-2), bla(IMP-1) and bla(OXA-23) genes by using loop-mediated isothermal amplification methods.

Kim HJ, Kim HS, Lee JM, Yoon SS, Yong D - Ann Lab Med (2016)

Determination of the detection limit of the loop-mediated isothermal amplification (LAMP) assays and the minimum reaction time. (A-C) Various amounts of template DNA (1 fg, 10 fg, 100 fg, 1 pg, 10 pg, 100 pg, 1 ng, or 10 ng) were used in the LAMP reactions. The amplification was performed with 2mM MgSO4 at 62℃ for 1 hr. (D-F) LAMP reactions were performed for a range of reaction times. After the reaction time indicated on top of the gels, the LAMP reaction was terminated by inactivating Bst DNA polymerase by incubation at 95℃ for 3 min. The reaction products were loaded onto a 1.5% agarose gel for analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697338&req=5

Figure 3: Determination of the detection limit of the loop-mediated isothermal amplification (LAMP) assays and the minimum reaction time. (A-C) Various amounts of template DNA (1 fg, 10 fg, 100 fg, 1 pg, 10 pg, 100 pg, 1 ng, or 10 ng) were used in the LAMP reactions. The amplification was performed with 2mM MgSO4 at 62℃ for 1 hr. (D-F) LAMP reactions were performed for a range of reaction times. After the reaction time indicated on top of the gels, the LAMP reaction was terminated by inactivating Bst DNA polymerase by incubation at 95℃ for 3 min. The reaction products were loaded onto a 1.5% agarose gel for analysis.
Mentions: To assess the limit of detection (LOD) for each LAMP reaction, LAMP assays were performed by using a series of DNA template samples prepared from 10-fold serial dilutions of a stock with an initial concentration of 10 ng/µL. A typical ladder-like pattern was detected by using as little as 1 pg of target DNA for blaVIM-2 and 10 pg of target DNA for blaIMP-1 and blaOXA-23 (Fig. 3A-C). We then compared the LOD for each target gene in conventional PCR.

Bottom Line: The optimal reaction temperature and incubation time were determined by using a gradient thermocycler.Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2).These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance.

Methods: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO₄) by testing different concentrations of MgSO₄. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates.

Results: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles.

Conclusions: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.

No MeSH data available.


Related in: MedlinePlus