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 degrees 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 degrees 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 micro l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 micro 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.

FIG. 1.

(A) Schematic representation of primers used in this study. Construction of the inner primers FIP and BIP is shown. F1c and B2c, complementary sequences of F1 and B2, respectively. (B) Nucleotide sequences of gyrB and 16S rDNA used for designing the primers. Recognition sequences of the primers are shown between capital letters. A right arrow indicates that a sense sequence is used for the primer. A left arrow indicates that a complementary sequence is used for the primer.

FIG. 2.

(A) Visual inspection and electrophoretic analysis of LAMP amplified products. Lane M, 100-bp ladder used as a size marker; lanes 1, 3, and 4, LAMP carried out with MTB primers in the presence of genomic DNA from M. tuberculosis, M. avium, and M. intracellulare, respectively; lane 2, LAMP product from lane 1 after digestion with BsaI; lanes 5, 10, and 15, LAMP carried out in the absence of template DNA with MTB, MAV, and MIN primers, respectively; lanes 6, 8, and 9, LAMP carried out with MAV primers in the presence of genomic DNA from M. avium, M. tuberculosis, and M. intracellulare, respectively; lane 7, LAMP product from lane 6 after digestion with HaeIII; lanes 11, 13, and 14, LAMP carried out with MIN primers in the presence of genomic DNA from M. intracellulare, M. tuberculosis, and M. avium, respectively; lane 12, LAMP product from lane 11 after digestion with Tth111I. (B) Sensitivities of visual inspection and electrophoretic analysis of LAMP amplified products. The number above each tube or lane represents the dilution of the LAMP product: ×1, no dilution; ×5, 5-fold dilution; ×15, 15-fold dilution; ×30, 30-fold dilution; ×50, 50-fold dilution; ×100, 100-fold dilution.