Loop-mediated isothermal amplification for detection of African trypanosomes

Abstract

While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.

FIG. 1.

(A) Schematic presentation of a double-stranded target DNA (solid lines) and LAMP inner (FIP and BIP) and outer (F3 and B3) primer pairs (open boxes). The FIP (BIP) primer consists of F1c (or B1c), a TTTT spacer (dotted line), and F2 (B2). (B) Nucleic acid sequence of minimum PFR A-specific LAMP (primer set A1, see Table 1) reaction unit. Two inverted repeats are indicated by solid arrows and dotted arrows. FIP and BIP primers are indicated below the sequence as < FIP > and < BIP >, respectively. A probe used for Southern blot analysis of LAMP products is designed to hybridize the region indicated by dotted line. (C) Schematic presentation of the single-stranded minimum LAMP reaction unit. Inverted repeats at both ends (solid and dotted lines) of the fragment form stem-loop structures. A probe used for Southern blot analysis of LAMP products is designed to hybridize the region indicated by the dotted line.

FIG. 2.

LAMP reactions for T. brucei and T. congolense. Four sets of primers were designed to hybridize to the gene encoding T. brucei PFR A (A1 and A2) and T. congolense ribosomal subunit protein P0 (P01 and P02). The LAMP products were electrophoresed in 1.5% agarose gel and stained with ethidium bromide. Template DNAs were obtained from T. brucei GUTat 3.1 (B) and T. congolense IL-3000 (C). Size markers (1-kbp ladder) were electrophoresed in lane M, and their sizes are indicated on the left. Lanes N and P, negative and positive reaction controls, respectively.

FIG. 3.

Comparison of detection sensitivity in LAMP and PCR (A). Total DNAs from T. brucei GUTat 3.1 and T. congolense IL-3000 were serially diluted from 10 ng to 1 pg and amplified by LAMP and PCR. A1 and P01 are primer sets used in the LAMP reactions. The F3 and B3 primers in each LAMP primer set were used in the PCR. The sizes of the 1-kb size markers in lane M are indicated on the left. (B) Southern blot analyses of the LAMP products. The same LAMP and PCR products shown in A were probed with the synthetic oligonucleotide probes. The probes do not hybridize to either inner (FIP and BIP) or outer (F3 and B3) primer binding regions, as shown in Fig. 1B and C.

FIG. 4.

Characterization of a different amplification pattern of PFR A-specific LAMP. (A) Total DNA from T. brucei GUTat 3.1 was serially diluted from 10 ng to 1 pg and amplified by PFR A-specific LAMP. PFR A A1 primer sets were used in the LAMP reactions. The band pattern of lane 4* is different from the others (lanes 1, 2, 3, and 5). The sizes of the 1-kb size markers in lane M are indicated on the left. (B) Comparison of nucleic acid sequences between the regular LAMP product (LAMP) and that of lane 4* (Clone 1). Sequence features are described between the < and > signs. BIPc indicates the complementary strand of the BIP primer. Insertions and deletions found in clone 1 are indicated by asterisks and hyphens, respectively.

FIG. 5.

Sequential analysis of blood samples obtained from mouse 1 infected with T. brucei gambiense IL-3253. The samples were examined by microscopic observation of buffy coat samples, PFR A-specific LAMP with primer set A1, and PFR A-specific PCR with primers F3 and B3 of the A1 primer set. PCRs were performed under enhanced conditions as described in Materials and Methods. Numbers above each lane indicate days postinfection (DPI). + indicates the presence of trypanosomes in buffy coat samples observed by microscopy. The sizes of the markers in lane M are indicated on the left.