Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients

Abstract

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 within 15 min, with only two manipulation/pipetting steps needed. The diagnostic sensitivity and specificity of this extraction protocol together with M. ulcerans RPA in comparison with standard DNA extraction with real-time PCR was 87% (n = 26) and 100% (n = 13), respectively. We have established a simple, fast and efficient protocol for the extraction and detection of M. ulcerans DNA in clinical samples that is adaptable to field conditions.

A combined effort of 11 laboratories in the WHO African region to improve quality of Buruli ulcer PCR diagnosis: The “BU-LABNET”

“…From the 11 SOPs received by CPC, we listed 4 main methods of DNA extraction used: 1) Automatized DNA extraction using the Maxwell Instrument (Promega), used by 2 laboratories: 2) Commercial kits; Gentra Puregene kit (as stated in the Buruli ulcer Manual for Health Care Providers) and QIAamp DNA mini kit with an enzymatic lysis step (Qiagen), used by 4 laboratories: 3) Alkaline lysis method using “in-house” buffers [ 8 ] and a commercial kit [ 9 ] (GenoLyse, Hain LifeScience) used by 4 other laboratories, and 4) The Modified Boom method [ 10 ] used by a single laboratory.…”

Comparative Performance of Line Probe Assay and GeneXpert in the Detection of Rifampicin Monoresistance in a TB-Endemic African Country

“…Kappa statistics were used to assess degree of agreement between GeneXpert and LPA with DST culture in detecting rifampicin-resistant TB. The receiver operating curve (ROC) was used to determine discriminatory ability of tests in distinguishing resistant and susceptible TB isolate [ 28 , 31 ].…”

Point-Of-Care or Point-Of-Need Diagnostic Tests: Time to Change Outbreak Investigation and Pathogen Detection

“…A quick extraction method, which incorporates detergent, proteinases, and heat, was used to obtain the RNA from hepatitis C [ 40 ] and Ebola viruses [ 41 ]. Another approach based on two steps of alkaline lysis then neutralization was used for DNA collection from skin samples of Buruli ulcer patients [ 42 ]. Despite the availability of rapid extraction techniques, so far, they have not been widely used.…”