scholarly journals A multiplex loop-mediated isothermal amplification assay for rapid screening of Acinetobacter baumannii and D carbapenemase OXA-23 gene

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Rungong Yang ◽  
Honghong Zhang ◽  
Xiaoxia Li ◽  
Ling Ye ◽  
Meiliang Gong ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acinetobacter Baumannii ◽  
Resistance Gene ◽  
Traditional Method ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Reca Gene ◽  
Drug Resistance Gene ◽  
Loop Mediated Isothermal Amplification

Background: Acinetobacter baumannii is a health burden responsible for various nosocomial infections, and bacteremia in particular. The resistance of A. baumannii to most antibiotics including carbapenem has increased. OXA-23-producing A. baumannii is the chief source of nosocomial outbreaks with carbapenem-resistant A. baumannii. Successful antibiotic treatment relies on the accurate and rapid identification of infectious agents and drug resistance. Here, we describe a multiplex loop-mediated isothermal amplification (LAMP) assay for simultaneous and homogeneous identification for A. baumannii infection screening and drug-resistance gene detection. Methods: Four primer pairs were designed to amplify fragments of the recA gene of A. baumannii and the oxa-23 gene. The reaction with a 25 μl of final volume was performed at 63°C for 60 min. For comparative purposes, we used a traditional method of bacterial identification to evaluate assay efficacy. Results: The multiplex LAMP assay enables simultaneous and homogeneous detection of the recA gene of A. baumannii and the oxa-23 gene and requires less than 21 min with no pre-requisite for DNA purification prior to the amplification reaction. The detection is specific to A. baumannii, and the coincidence rate of the multiplex LAMP and the traditional method was 100%. Conclusions: Our data indicate that the multiplex LAMP assay is a rapid, sensitive, simultaneous and homogeneous method for screening of A. baumannii and its drug-resistance gene.

scholarly journals Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus–baumannii Complex and Associated Carbapenem Resistance

2021 ◽  
Vol 8 ◽  
Author(s):  
Amit Sharma ◽  
Rajni Gaind
Keyword(s):  
Acinetobacter Baumannii ◽  
Lamp Assay ◽  
Carbapenem Resistance ◽  
Acinetobacter Calcoaceticus ◽  
Isothermal Amplification ◽  
Colony Count ◽  
Dna Concentration ◽  
Loop Mediated Isothermal Amplification ◽  
Carbapenem Resistant ◽  
Clinically Significant

Background:Acinetobacter calcoaceticus–baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by blaOXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by blaOXA-23.Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S–23S rRNA and blaOXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR).Results: The optimal temperature for the LAMP assay was 65°C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg/μl and 1 ng/μl of DNA concentration and 104 cfu/ml and 108 cfu/ml of colony count, respectively. The LAMP assay was 10- and 104-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species.Significance of the study: The LAMP assay can be applied for early detection of significant species of the ACB complex from clinical samples and their carbapenem-resistant variants. Depending on the emerging pathogen and locally prevalent resistance genes, the LAMP assay can be modified for detection of colonization or infection by various resistant bugs.

scholarly journals Development of a loop-mediated isothermal amplification assay combined with a nanoparticle-based lateral flow biosensor for rapid detection of plasmid-mediated colistin resistance gene mcr-1

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249582
Author(s):  
Lin Gong ◽  
Fei Tang ◽  
Ernan Liu ◽  
Xiaoli Liu ◽  
Huiqiong Xu ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Lamp Assay ◽  
High Specificity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Reaction Products ◽  
Colistin Resistance ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Amplification Assay

A loop-mediated isothermal amplification assay combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) was established for the rapid and accurate detection of the mobilized colistin resistance gene (mcr-1), which causes the loss of colistin antibacterial efficacy in clinical treatments. The amplification stage of the assay was completed in 60 min at 63°C, and the reaction products could be visually detected by employing the LFB, which provided a fast (within 2 min) and objective method to evaluate the amplification results. The LAMP assay amplified the target sequences of mcr-1 with high specificity. In pure strains, the detection limit of the LAMP-LFB assay was 360 fg plasmid DNA/reaction, and in spiked feces samples the value was approximately 6.3×103 CFU/mL (~6.3 CFU/reaction), which was tenfold more sensitive than the PCR assay. The results show that the developed LAMP-LFB assay will be a worthy tool for the simple, rapid, specific, and sensitive detection of mcr-1 gene in clinical settings and resource-limited areas.

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