Recombinase polymerase amplification assay for detection of Mycobacterium ulcerans DNA v1 (original) (raw)
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protocols.io, 2018
This document describes the standard operating procedure for the application of the real time Mu-RPA assay with Exo probe system. The assay detects M. ulcerans DNA (IS2404) from clinical sample or culture suspension. The assay consists of recombinase polymerase amplification TwistDx Exo kit procedure. The reaction mix must be prepared in an environment free of DNA amplicons. Personal protective clothing (i.e. lab coats, gloves) must be used throughout the process.
Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay
PLOS Neglected Tropical Diseases, 2019
Background Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42˚C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95).
Development and Application of Real-Time PCR Assay for Quantification of Mycobacterium ulcerans DNA
Journal of Clinical Microbiology, 2003
Buruli ulcer, an infection caused by Mycobacterium ulcerans, is, after tuberculosis and leprosy, the third most common mycobacterial disease. The mode of transmission of M. ulcerans is not exactly known, but since Buruli ulcer often occurs in focalized swampy areas, it is assumed that there is a reservoir of the pathogen in stagnant water. Buruli ulcer usually starts as a painless nodule and can lead to massive destruction of skin, subcutaneous tissue, and eventually muscle and bone. Currently the only recommended treatment is wide surgical excision. In this report we describe the development of a real-time PCR method for the quantification of M. ulcerans DNA (IS2404 TaqMan). The highly specific assay is based on the detection of the M. ulcerans specific insertion sequence IS2404. The IS2404 TaqMan assay turned out to be about 10 times more sensitive than the available conventional PCR-based diagnostic test. It is demonstrated that the IS2404 TaqMan assay is suitable for the quantitative assessment of the dissemination of the mycobacteria in Buruli ulcer lesions. Prototype results obtained with excised tissue from a patient with a late preulcerative Buruli ulcer lesion reconfirmed earlier histopathological findings indicating that tissue damage occurs far beyond the regions in which large numbers of mycobacteria are detectable. The IS2404 TaqMan assay should be a useful tool for both diagnosis and research into the pathology and mode of transmission of this still inadequately investigated mycobacterial disease.
Development of a PCR assay for rapid diagnosis of Mycobacterium ulcerans infection
Journal of clinical microbiology, 1997
The diagnosis of Mycobacterium ulcerans infection is hampered by the slow growth of the bacterium in culture, resulting in a delay of several months before a specific diagnosis can be obtained. In addition, M. ulcerans cannot be isolated from water even when there is convincing epidemiological evidence implicating this as the source of infection. The aim of the present study was to develop a PCR assay to circumvent the problems of delayed diagnosis and insensitivity of standard bacterial culture for M. ulcerans. For the PCR, we isolated an M. ulcerans-specific DNA fragment, 1,109 bp long, which is repeated at least 50 times throughout the genome. Use of this sequence as a target for PCR allowed us to detect as few as 2 molecules of genomic DNA in vitro. The PCR was used to detect M. ulcerans DNA in fresh tissue and paraffin-embedded sections from all seven patients with culture-confirmed cases of infection.
2019
Introduction: Buruli ulcer (BU) is a serious skin disease caused by Mycobacterium ulcerans. According to WHO, 70% of BU cases should be confirmed by the PCR-IS2404 gene amplification. The objective of this study is to validate a real-time PCR for the detection of M. ulcerans in clinical and environmental samples. Methodology: A total of 70 clinical samples, 10 M. ulcerans strains and 15 environmental samples were tested by Ziehl-Neelsen staining technique, conventional PCR, real time qPCR and culture. The proportion of positive cases of M. ulcerans detection between the different tests was compared by the Chi-square test. The difference was statistically significant for a P-value ≤ 0.05. Results: Out of 33/80 samples were cultured positive (41.25%) to M. ulcerans, 41/80 ZN staining were positive (51.25%) to AFB at the microscopy, 55/80 (68.75%) and 64/80 samples (80%) were positive to the IS2404 insertion sequence from conventional PCR and qPCR respectively. Both PCR techniques show...
Journal of clinical microbiology, 1997
We compared various diagnostic tests for their abilities to detect Mycobacterium ulcerans infection in specimens from patients with clinically active disease. Specimens from 10 patients from the area of Zangnanado (Department of Zou, Benin) with advanced, ulcerated active M. ulcerans infections were studied by direct smear, histopathology, culture, PCR, and oligonucleotide-specific capture plate hybridization (OSCPH). A total of 27 specimens, including 12 swabs of exudate collected before debridement and 15 fragments of tissue obtained during debridement, were submitted to bacteriologic and histopathologic analysis. The histopathologic evaluation of tissues from all six patients so tested revealed changes typical of those caused by M. ulcerans infection. Five specimens were contaminated, and M. ulcerans was cultivated on Löwenstein-Jensen medium from 12 of the remaining 22 (54.5%) specimens. Detection of mycobacteria was performed by PCR, and M. ulcerans was detected by OSCPH with a...
A quick and cost effective method for the diagnosis of Mycobacterium ulcerans infection
BMC Infectious Diseases, 2012
Background: Buruli ulcer (BU), a neglected tropical skin disease caused by Mycobacterium ulcerans, has been reported in over 30 countries worldwide and is highly endemic in rural West and Central Africa. The mode of transmission remains unknown and treatment is the only alternative to disease control. Early and effective treatment to prevent the morbid effects of the disease depends on early diagnosis; however, current diagnosis based on clinical presentation and microscopy has to be confirmed by PCR and other tests in reference laboratories. As such confirmed BU diagnosis is either late, inefficient, time consuming or very expensive, and there is the need for an early diagnosis tool at point of care facilities. In this paper we report on a simple, quick and inexpensive diagnostic test that could be used at point of care facilities, in resource-poor settings.
Diagnostics
Isothermal amplification techniques such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for diagnosing Buruli ulcer, a necrotic skin disease caused by Mycobacterium ulcerans, have renewed hope for the molecular diagnosis of clinically suspected Buruli ulcer cases in endemic districts. If these techniques are applied at district-level hospitals or clinics, they will help facilitate early case detection with prompt treatment, thereby reducing disability and associated costs of disease management. The accuracy as well as the application of these molecular techniques at point of need is dependent on simple and fast DNA extraction. We have modified and tested a rapid extraction protocol for use with an already developed recombinase polymerase amplification assay. The entire procedure from “sample in, extraction and DNA amplification” was conducted in a mobile suitcase laboratory within 40 min. The DNA extraction procedure was performed wit...
International journal of tropical diseases, 2020
Background: Conventional and nested polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP) have been used to identify Mycobacterium ulcerans in separate studies and different specimens. However, the sensitivities of these three techniques have not been compared in a single study. Objective: This study compared the performance of two variant PCR techniques and LAMP assays to detect M. Ulcerans in same clinical specimens. Methods: Samples were collected from patients suspected of Buruli ulcer disease (BUD) in Southern Ghana. Ulcerative and non-ulcerative forms of the disease were swabbed and aspirated respectively. Insertion sequence 2404 (IS2404) M. ulcerans targets were detected in each sample using conventional polymerase chain reaction (PCR), nested PCR and loop-mediated isothermal amplification (LAMP) assay. Results: In all, 141 suspected BUD patients were sampled (Amasaman, n = 52; Obom, n = 17; Paakro, n = 31; Nkawie, n = 21 and Tepa, n = 20). The reference technique, nested PCR, detected M. ulcerans in 122 (86.5%) whereas conventional PCR and BU-LAMP detected M. ulcerans in 104 (73.7%) and 119 (84.4%) samples respectively. Compared to nested PCR, conventional PCR performed poorly (x 2 = 19.7, p < 0.01; κ = 0.58; % agreement = 86.62) while BU-LAMP was as good as nested PCR (x 2 = 0.457, p = 0.459; κ = 0.88; % agreement = 97.16). Sensitivity of conventional PCR and BU-LAMP were 84.6% (95% CI: 76.9-90.4) and 97.5% (95% CI: 92.9-99.5) respectively. BU-LAMP and nested PCR detected M. ulcerans in ulcerative forms of the disease and category II lesions better while the three techniques did not differ in sensitivities in other clinical forms and lesion categories. Conclusion: BU-LAMP assay is very comparable to nested PCR in detecting M. ulcerans from clinical specimens. More importantly, LAMP assay is user-friendly, fast, requires less instrumentation and easy to use in resource-limited laboratory with minimal user training.
Journal of Microbiological Methods, 2009
Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell® 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h 157 L. Durnez et al. / Journal of Microbiological Methods 76 (2009) 152-158