Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis

Abstract

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.

FIG. 1.

Design of LAMP primers.

FIG. 2.

Positions of LAMP primers on IS900.

FIG. 3.

Detection of turbidity for the LAMP method. Left tube, negative; right tube, positive.

FIG. 4.

(A) Turbidity measurements of LAMP reaction mixtures for MAP detection using P-1. (B) Electrophoretic analysis of LAMP reaction mixtures for MAP detection using P-1. Lane 1, 5 pg of MAP per μl; lane 2, 0.5 pg of MAP per μl; lane 3, 500 pg of strain number 51 per μl; lane 4, 50 pg of strain number 51 per μl; lane 5, 500 pg of strain number 52 per μl; lane 6, 50 pg of strain number 52 per μl; lane 7, 0.05 pg of plasmid DNA of IS900 per μl; lane 8, negative control; lane M, 100-bp ladder. Abs., absorbance.

FIG. 5.

(A) Turbidity measurements of LAMP reaction mixtures for MAP detection using P-2. (B) Electrophoretic analysis of LAMP reaction mixtures for MAP detection using P-2. Lane 1, 50 pg of MAP per μl; lane 2, 20 pg of MAP per μl; lane 3, 10 pg of MAP per μl; lane 4, 5 pg of MAP per μl; lane 5, 1 pg of MAP per μl; lane 6, 0.1 pg of plasmid DNA per μl; lane 7, negative control; lane M, 100-bp ladder. Abs., absorbance.

FIG. 6.

Restriction analysis of LAMP products using P-1 (A) and P-2 (B). (A) Lane 1, amplified products of pIS900; lane 2, pIS900 digested with AflIII; lane 3, amplified products of MAP; lane 4, MAP digested with AflIII. (B) Lane 1, amplified products of pIS900; lane 2, pIS900 digested with PvuII; lane 3, amplified products of MAP; lane 4, MAP digested with PvuII; lane M, 100-bp ladder.