Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium , and M. intracellulare in Sputum Samples

Abstract

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63°C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.Because of their slow growth rate, identification of mycobacteria is a notorious problem for public health and clinical laboratories. To address the need for rapid and sensitive identification of Mycobacterium tuberculosis and other mycobacteria, various genotyping methods for routine diagnosis have been introduced during the past decade (1,2,4,9,10,(21)(22)(23)25). The recent trend in genetic testing is to make systems fully automatic with high-throughput analysis. Although this may be an ideal approach, it requires expensive equipment as well as a huge amount of space in routine diagnostic laboratories. As a result, it centralizes genetic testing in highly sophisticated facilities. If this trend continues, escalated pressure for transportation of clinical specimens will pose another problem. On the other hand, there are pressing needs for point-of-care testing at hospitals and primary care facilities.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method which relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment (13,(15)(16)(17)...

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

“…For this purpose, the buffer, primers and DNA polymerase are mixed together and the mixture is incubated at 65 8C in a regular laboratory water bath or heat block that provides a constant temperature. The specificity of the assay is extremely high because it uses four primers for the recognition of six distinct regions on the target DNA (Notomi et al, 2000;Iwamoto et al, 2003). The LAMP assay was initially evaluated for detection of hepatitis B virus DNA (Notomi et al, 2000), and has recently been applied to the direct detection of the Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare (Iwamoto et al, 2003), and severe acute respiratory syndrome coronavirus (Hong et al, 2004).…”

Rapid detection of Mycobacterium tuberculosis complex in cattle and lechwe (Kobus leche kafuensis) at the slaughter house

“…These results clearly demonstrate the high sensitivity of the LAMP system in detecting TB as observed by others who have been working on human specimens. 11,17,21 This result also suggests that LAMP may be superior to all procedures being used at the moment to detect TB in animals. One sample was detected as positive on LAMP although negative on culture.…”

Non Specific Amplification with the LAMP Technique in the Diagnosis of Tuberculosis in Sri Lankan Settings

“…One assay is to identify Mycobacterium genus using Mycobacterium universal (Muniv) primers [8]. The other two assays used MTB specific primers targeting rimM gene sequence [9] or gyrB gene sequence [8], which enabled detection up to the species level. A culture of standard MTB strain, H37Rv, was used as a positive control.…”