Abstract
BackgroundTuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease.Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries.MethodsIn the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process.To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB.ResultsOur SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA.ConclusionTo our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.
Highlights
Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries
When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-Loop-mediated isothermal amplification (LAMP) assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards
In order to determine whether our single step LAMP (SS-LAMP) assay can be performed in a single step procedure, we amplified the insertion sequence 6110 (IS6110) target sequence directly from the liquefied sputum samples
Summary
Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. In Morocco, as in low and middle income countries, the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD) recommend the diagnosis of TB by sputum smear microscopy as a crucial and inevitable step in testing patients suspected of having pulmonary TB [2, 3]. This technique seems to be simple, rapid and inexpensive but has the inconvenience to be less sensitive and requires repeated patient’s sputum examinations [3]
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