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Research article
Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae
Aleksandra Anna Zasada
National Institute of Public Health - National Institute of Hygiene
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2774-0941
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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.
Keywords
LAMP, POCT, diphtheria, Corynebacterium diphtheriae, colorimetric detection, lateral flow dipstick
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CURRENT STATUS: Under Review
Version 5
Posted 15 Apr, 2020
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Editor assigned
On 12 Apr, 2020
Submission checks complete
On 11 Apr, 2020
Editor invited
On 11 Apr, 2020
No comments provided
Submission checks complete
On 06 Apr, 2020
Editorial decision: Minor revision
On 06 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 03 Apr, 2020
Editor assigned
On 15 Mar, 2020
Submission checks complete
On 14 Mar, 2020
Editor invited
On 14 Mar, 2020
No comments provided
Editorial decision: Minor revision
On 03 Mar, 2020
Review #1 received
Received 21 Feb, 2020
Review #2 received
Received 18 Feb, 2020
Reviewer #3 agreed
On 11 Feb, 2020
Reviewer #2 agreed
On 10 Feb, 2020
Editor assigned
On 07 Feb, 2020
Reviewers invited
Invitations sent on 07 Feb, 2020
Reviewer #1 agreed
On 07 Feb, 2020
Submission checks complete
On 06 Feb, 2020
Editor invited
On 06 Feb, 2020
No comments provided
Editorial decision: Major revision
On 31 Dec, 2019
Review #1 received
Received 05 Dec, 2019
Review #4 received
Received 27 Nov, 2019
Review #2 received
Received 18 Nov, 2019
Review #3 received
Received 18 Nov, 2019
Reviewer #4 agreed
On 15 Nov, 2019
Reviewer #3 agreed
On 13 Nov, 2019
Reviewer #1 agreed
On 12 Nov, 2019
Reviewer #2 agreed
On 12 Nov, 2019
Reviewers invited
Invitations sent on 18 Oct, 2019
Editor assigned
On 27 Sep, 2019
Submission checks complete
On 20 Sep, 2019
Editor invited
On 20 Sep, 2019
First submitted
On 17 Sep, 2019
Metrics
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Subject Areas
Infectious Diseases
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This preprint is under consideration at BMC Infectious Diseases. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae
Aleksandra Anna Zasada
National Institute of Public Health - National Institute of Hygiene
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2774-0941
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 5
Posted 15 Apr, 2020
No community comments so far
Editor assigned
On 12 Apr, 2020
Submission checks complete
On 11 Apr, 2020
Editor invited
On 11 Apr, 2020
No comments provided
Submission checks complete
On 06 Apr, 2020
Editorial decision: Minor revision
On 06 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 03 Apr, 2020
Editor assigned
On 15 Mar, 2020
Submission checks complete
On 14 Mar, 2020
Editor invited
On 14 Mar, 2020
No comments provided
Editorial decision: Minor revision
On 03 Mar, 2020
Review #1 received
Received 21 Feb, 2020
Review #2 received
Received 18 Feb, 2020
Reviewer #3 agreed
On 11 Feb, 2020
Reviewer #2 agreed
On 10 Feb, 2020
Editor assigned
On 07 Feb, 2020
Reviewers invited
Invitations sent on 07 Feb, 2020
Reviewer #1 agreed
On 07 Feb, 2020
Submission checks complete
On 06 Feb, 2020
Editor invited
On 06 Feb, 2020
No comments provided
Editorial decision: Major revision
On 31 Dec, 2019
Review #1 received
Received 05 Dec, 2019
Review #4 received
Received 27 Nov, 2019
Review #2 received
Received 18 Nov, 2019
Review #3 received
Received 18 Nov, 2019
Reviewer #4 agreed
On 15 Nov, 2019
Reviewer #3 agreed
On 13 Nov, 2019
Reviewer #1 agreed
On 12 Nov, 2019
Reviewer #2 agreed
On 12 Nov, 2019
Reviewers invited
Invitations sent on 18 Oct, 2019
Editor assigned
On 27 Sep, 2019
Submission checks complete
On 20 Sep, 2019
Editor invited
On 20 Sep, 2019
First submitted
On 17 Sep, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Infectious Diseases
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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