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Rapid detection of Acinetobacter baumannii and molecular epidemiology of carbapenem-resistant A. baumannii in two comprehensive hospitals of Beijing, China.

Li P, Niu W, Li H, Lei H, Liu W, Zhao X, Guo L, Zou D, Yuan X, Liu H, Yuan J, Bai C - Front Microbiol (2015)

Bottom Line: The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR.In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research.We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Diseases, 307th Hospital of Chinese People's Liberation Army Beijing, China.

ABSTRACT
Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections. A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A. baumannii. Group 2 carbapenems have been considered the agents of choice for the treatment of multiple drug-resistant A. baumannii. But the prevalence of carbapenem-resistant A. baumannii (CRAB) has been steadily increasing in recent years. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of A. baumannii in clinical samples by using high-specificity primers of the bla OXA-51 gene. Then we investigated the OXA-carbapenemases molecular epidemiology of A. baumannii isolates in two comprehensive hospitals in Beijing. The results showed that the LAMP assay could detect target DNA within 60 min at 65°C. The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR. Furthermore, this method could distinguish A. baumannii from the homologous A. nosocomialis and A. pittii. A total of 228 positive isolates were identified by this LAMP-based method for A. baumannii from 335 intensive care unit patients with clinically suspected multi-resistant infections in two hospitals in Beijing. The rates of CRAB are on the rise and are slowly becoming a routine phenotype for A. baumannii. Among the CRABs, 92.3% harbored both the bla OXA-23 and bla OXA-51 genes. Thirty-three pulsotypes were identified by pulsed-field gel electrophoresis, and the majority belonged to clone C. In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research. We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

No MeSH data available.


Related in: MedlinePlus

Sensitivity of the LAMP reaction and PCR for detection of the blaOXA-51-like gene. Genomic DNA was diluted in a set of serial 10-fold dilutions. Both LAMP reactions (A,B) and PCR (C) were performed in duplicate for each dilution. Tubes and lanes: 1: 500 ng/μl, 2: 50 ng/μl, 3: 5 ng/μl, 4: 500 pg/μl, 5: 50 pg/μl, 6: 5 pg/μl, 7: 0.5 pg/μl, 8: 0.05 pg/μl. (A) Turbidity was monitored using a Loopamp real-time turbidimeter by measuring the absorbance at 650 nm every 6 s. (B) The direct visual method for the detection of LAMP. One microliter of fluorescent detection reagent was added to 25 μl of LAMP reaction mixture before the LAMP reactions were initiated. (C) PCR products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide.
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Figure 4: Sensitivity of the LAMP reaction and PCR for detection of the blaOXA-51-like gene. Genomic DNA was diluted in a set of serial 10-fold dilutions. Both LAMP reactions (A,B) and PCR (C) were performed in duplicate for each dilution. Tubes and lanes: 1: 500 ng/μl, 2: 50 ng/μl, 3: 5 ng/μl, 4: 500 pg/μl, 5: 50 pg/μl, 6: 5 pg/μl, 7: 0.5 pg/μl, 8: 0.05 pg/μl. (A) Turbidity was monitored using a Loopamp real-time turbidimeter by measuring the absorbance at 650 nm every 6 s. (B) The direct visual method for the detection of LAMP. One microliter of fluorescent detection reagent was added to 25 μl of LAMP reaction mixture before the LAMP reactions were initiated. (C) PCR products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide.

Mentions: To compare the detection limit of traditional PCR with that of LAMP using either real-time turbidity or color-change measurements, 10-fold serial dilutions (50 ng/μl–5 pg/μl) were tested using genomic DNA extracted from A. baumannii ATCC 22933. As shown in Figure 4, the detection limits of real-time turbidity and visual detection were both 50 pg/μl, which was 10-fold more sensitive than traditional PCR assay.


Rapid detection of Acinetobacter baumannii and molecular epidemiology of carbapenem-resistant A. baumannii in two comprehensive hospitals of Beijing, China.

Li P, Niu W, Li H, Lei H, Liu W, Zhao X, Guo L, Zou D, Yuan X, Liu H, Yuan J, Bai C - Front Microbiol (2015)

Sensitivity of the LAMP reaction and PCR for detection of the blaOXA-51-like gene. Genomic DNA was diluted in a set of serial 10-fold dilutions. Both LAMP reactions (A,B) and PCR (C) were performed in duplicate for each dilution. Tubes and lanes: 1: 500 ng/μl, 2: 50 ng/μl, 3: 5 ng/μl, 4: 500 pg/μl, 5: 50 pg/μl, 6: 5 pg/μl, 7: 0.5 pg/μl, 8: 0.05 pg/μl. (A) Turbidity was monitored using a Loopamp real-time turbidimeter by measuring the absorbance at 650 nm every 6 s. (B) The direct visual method for the detection of LAMP. One microliter of fluorescent detection reagent was added to 25 μl of LAMP reaction mixture before the LAMP reactions were initiated. (C) PCR products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585070&req=5

Figure 4: Sensitivity of the LAMP reaction and PCR for detection of the blaOXA-51-like gene. Genomic DNA was diluted in a set of serial 10-fold dilutions. Both LAMP reactions (A,B) and PCR (C) were performed in duplicate for each dilution. Tubes and lanes: 1: 500 ng/μl, 2: 50 ng/μl, 3: 5 ng/μl, 4: 500 pg/μl, 5: 50 pg/μl, 6: 5 pg/μl, 7: 0.5 pg/μl, 8: 0.05 pg/μl. (A) Turbidity was monitored using a Loopamp real-time turbidimeter by measuring the absorbance at 650 nm every 6 s. (B) The direct visual method for the detection of LAMP. One microliter of fluorescent detection reagent was added to 25 μl of LAMP reaction mixture before the LAMP reactions were initiated. (C) PCR products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide.
Mentions: To compare the detection limit of traditional PCR with that of LAMP using either real-time turbidity or color-change measurements, 10-fold serial dilutions (50 ng/μl–5 pg/μl) were tested using genomic DNA extracted from A. baumannii ATCC 22933. As shown in Figure 4, the detection limits of real-time turbidity and visual detection were both 50 pg/μl, which was 10-fold more sensitive than traditional PCR assay.

Bottom Line: The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR.In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research.We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Diseases, 307th Hospital of Chinese People's Liberation Army Beijing, China.

ABSTRACT
Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections. A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A. baumannii. Group 2 carbapenems have been considered the agents of choice for the treatment of multiple drug-resistant A. baumannii. But the prevalence of carbapenem-resistant A. baumannii (CRAB) has been steadily increasing in recent years. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of A. baumannii in clinical samples by using high-specificity primers of the bla OXA-51 gene. Then we investigated the OXA-carbapenemases molecular epidemiology of A. baumannii isolates in two comprehensive hospitals in Beijing. The results showed that the LAMP assay could detect target DNA within 60 min at 65°C. The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR. Furthermore, this method could distinguish A. baumannii from the homologous A. nosocomialis and A. pittii. A total of 228 positive isolates were identified by this LAMP-based method for A. baumannii from 335 intensive care unit patients with clinically suspected multi-resistant infections in two hospitals in Beijing. The rates of CRAB are on the rise and are slowly becoming a routine phenotype for A. baumannii. Among the CRABs, 92.3% harbored both the bla OXA-23 and bla OXA-51 genes. Thirty-three pulsotypes were identified by pulsed-field gel electrophoresis, and the majority belonged to clone C. In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research. We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

No MeSH data available.


Related in: MedlinePlus