Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae
Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.
Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.
Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.
Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.
Figure 1
Figure 2
Figure 3
Posted 15 Apr, 2020
On 12 Apr, 2020
On 11 Apr, 2020
On 11 Apr, 2020
On 06 Apr, 2020
On 06 Apr, 2020
On 03 Apr, 2020
On 15 Mar, 2020
On 14 Mar, 2020
On 14 Mar, 2020
On 03 Mar, 2020
Received 21 Feb, 2020
Received 18 Feb, 2020
On 11 Feb, 2020
On 10 Feb, 2020
On 07 Feb, 2020
Invitations sent on 07 Feb, 2020
On 07 Feb, 2020
On 06 Feb, 2020
On 06 Feb, 2020
On 31 Dec, 2019
Received 05 Dec, 2019
Received 27 Nov, 2019
Received 18 Nov, 2019
Received 18 Nov, 2019
On 15 Nov, 2019
On 13 Nov, 2019
On 12 Nov, 2019
On 12 Nov, 2019
Invitations sent on 18 Oct, 2019
On 27 Sep, 2019
On 20 Sep, 2019
On 20 Sep, 2019
On 17 Sep, 2019
Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae
Posted 15 Apr, 2020
On 12 Apr, 2020
On 11 Apr, 2020
On 11 Apr, 2020
On 06 Apr, 2020
On 06 Apr, 2020
On 03 Apr, 2020
On 15 Mar, 2020
On 14 Mar, 2020
On 14 Mar, 2020
On 03 Mar, 2020
Received 21 Feb, 2020
Received 18 Feb, 2020
On 11 Feb, 2020
On 10 Feb, 2020
On 07 Feb, 2020
Invitations sent on 07 Feb, 2020
On 07 Feb, 2020
On 06 Feb, 2020
On 06 Feb, 2020
On 31 Dec, 2019
Received 05 Dec, 2019
Received 27 Nov, 2019
Received 18 Nov, 2019
Received 18 Nov, 2019
On 15 Nov, 2019
On 13 Nov, 2019
On 12 Nov, 2019
On 12 Nov, 2019
Invitations sent on 18 Oct, 2019
On 27 Sep, 2019
On 20 Sep, 2019
On 20 Sep, 2019
On 17 Sep, 2019
Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.
Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.
Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.
Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.
Figure 1
Figure 2
Figure 3