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Loop-mediated isothermal amplification assays for screening of bacterial integrons.

Yu G, Chen L, Lin CW, Li B, Cui H, Chen S, Miao J, Bian H, Chen D, Deng Y - Biol. Res. (2014)

Bottom Line: As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively.As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.

Results: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%.

Conclusions: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

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Schematic diagram of primers used in the LAMP assays. In detail, 6–8 distinct regions on every strand were used to design LAMP primers for the target gene. For the inner primers, the forward inner primer (FIP) consisted of the complementary sequence of F1 (F1c), a T-T-T-T linker and F2; the backward inner primer (BIP) consisted of the complementary sequence of B1 (B1c), a T-T-T-T linker and B2. The outer primers F3 and B3 located outside of the F2 and B2 regions, with loop primers LF and LB located beween F2 and F1 or B1 and B2, respectively. The scare bar is 10 nm.
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Fig1: Schematic diagram of primers used in the LAMP assays. In detail, 6–8 distinct regions on every strand were used to design LAMP primers for the target gene. For the inner primers, the forward inner primer (FIP) consisted of the complementary sequence of F1 (F1c), a T-T-T-T linker and F2; the backward inner primer (BIP) consisted of the complementary sequence of B1 (B1c), a T-T-T-T linker and B2. The outer primers F3 and B3 located outside of the F2 and B2 regions, with loop primers LF and LB located beween F2 and F1 or B1 and B2, respectively. The scare bar is 10 nm.

Mentions: As screening and detection of integrons were concerned, polymerase chain reaction (PCR) has been widely used. However, the requirement for PCR cycler machine and electrophoresis of PCR amplicons have restricted its further application, especially in clinical laboratory [2, 26]. In the latest decade, loop-mediated isothermal amplification (LAMP), as a novel nucleic acid amplification method, had been reported [27–29] and applied to the detection of various pathogenic organisms [26, 30–45]. This LAMP methodology relies on an auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment, with 4 or 6 primers recognizing 6–8 distinct regions of the target gene (Figure 1) and generating the loop-mediated amplification under isothermal conditions between 60-65°C [27–29]. Amplicons are mixtures of many different sizes of stem-loop DNAs containing several inverted repeats of the target sequence and cauliflower-like structures with multiple loops [30, 46]. In this study, LAMP assays on resistance integrons screening (including class 1, 2 and 3 integrons) were evaluated, optimized and further applied to the detection of a large scale of clinical isolates, with approximately 60 min required for the entire process.Figure 1


Loop-mediated isothermal amplification assays for screening of bacterial integrons.

Yu G, Chen L, Lin CW, Li B, Cui H, Chen S, Miao J, Bian H, Chen D, Deng Y - Biol. Res. (2014)

Schematic diagram of primers used in the LAMP assays. In detail, 6–8 distinct regions on every strand were used to design LAMP primers for the target gene. For the inner primers, the forward inner primer (FIP) consisted of the complementary sequence of F1 (F1c), a T-T-T-T linker and F2; the backward inner primer (BIP) consisted of the complementary sequence of B1 (B1c), a T-T-T-T linker and B2. The outer primers F3 and B3 located outside of the F2 and B2 regions, with loop primers LF and LB located beween F2 and F1 or B1 and B2, respectively. The scare bar is 10 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4222780&req=5

Fig1: Schematic diagram of primers used in the LAMP assays. In detail, 6–8 distinct regions on every strand were used to design LAMP primers for the target gene. For the inner primers, the forward inner primer (FIP) consisted of the complementary sequence of F1 (F1c), a T-T-T-T linker and F2; the backward inner primer (BIP) consisted of the complementary sequence of B1 (B1c), a T-T-T-T linker and B2. The outer primers F3 and B3 located outside of the F2 and B2 regions, with loop primers LF and LB located beween F2 and F1 or B1 and B2, respectively. The scare bar is 10 nm.
Mentions: As screening and detection of integrons were concerned, polymerase chain reaction (PCR) has been widely used. However, the requirement for PCR cycler machine and electrophoresis of PCR amplicons have restricted its further application, especially in clinical laboratory [2, 26]. In the latest decade, loop-mediated isothermal amplification (LAMP), as a novel nucleic acid amplification method, had been reported [27–29] and applied to the detection of various pathogenic organisms [26, 30–45]. This LAMP methodology relies on an auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment, with 4 or 6 primers recognizing 6–8 distinct regions of the target gene (Figure 1) and generating the loop-mediated amplification under isothermal conditions between 60-65°C [27–29]. Amplicons are mixtures of many different sizes of stem-loop DNAs containing several inverted repeats of the target sequence and cauliflower-like structures with multiple loops [30, 46]. In this study, LAMP assays on resistance integrons screening (including class 1, 2 and 3 integrons) were evaluated, optimized and further applied to the detection of a large scale of clinical isolates, with approximately 60 min required for the entire process.Figure 1

Bottom Line: As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively.As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection.

Results: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 μL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%.

Conclusions: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

Show MeSH