A versatile isothermal amplification assay for the detection of leptospires from various sample types

Abstract

Background: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral secY gene.

Results: Several reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure Leptospira culture was 2 × 10⁴ copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease.

Conclusions: In short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study.

Keywords: Clinical detection; Loop-mediated isothermal amplification; Vector surveillance; Leptospirosis.

Figure 1. Sensitivity test of the LAMP assay using genomic DNA of L. interrogans serovar Pomona. The LAMP reaction was performed at 65 °C for 40 min.

(A) Realtime turbidimetry result of the LAMP reaction. (B) Colorimetric observation based on calcein dye color change. (C) Agarose gel electrophoresis of LAMP reaction products. Tubes and lanes 1 to 5: 2 × 10⁷ (100 ng) to 2 × 10³ copies (0.01 ng) of genomic DNA per reaction, NC: no-template control. Red square box indicates reaction tubes with calcein color change.

Figure 2. Specificity test of the LAMP assay based on colorimetric changes in calcein dye. The LAMP reaction was performed at 65 °C for 40 min using 300 ng of genomic DNA each.

(A) Specificity test on different serovars of Leptospira. Tube 1: L. interrogans serovar Pomona, 2: L. interrogans serovar Serawak, 3: L. interrogans serovar Canicola, 4: L. interrogans serovar Djasiman, 5: L. interrogans serovar Autumnalis, 6: L. interrogans serovar Australis, 7: L. interrogans serovar Pyrogenes, 8: L. interrogans serovar Lai, 9: L. interrogans serovar Copenhageni, 10: L. interrogans serovar Icterohaemorrhagiae, 11: L. interrogans serovar Bataviae, 12: L. interrogans serovar Hebdomadis, 13: L. kirschneri serovar Grippotyphosa, 14: L. borgpetersenii serovar Hardjo Bovis 15: L. biflexa serovar Patoc, NC: no-template control. (B) Specificity test on non-leptospiral bacteria. Tube 1: L. interrogans Pomona, 2: S. maltophillia ATCC, 3: P. aeruginosa, 4: A. baumannii ATCC, 5: E. coli XL10G, 6: S. pyogenes S28, 7: MRSA, 8: E. faecalis 33420, 9: E. faecium 4867, 10: C. difficile, NC: no-template control. Red square box indicates reaction tubes with calcein color change.

Figure 3. Sensitivity test of the LAMP assay using DNA isolated from spiked blood samples. The LAMP reaction was performed at 65 °C for 30 min.

(A) Realtime turbidimetry result of the LAMP reaction. (B) Colorimetric observation based on calcein dye color change. Tubes 1 to 6: 1 × 10⁵ to 1 leptospires/ml, tube 7: non-spiked blood, tube 8: no-template control. Red square box indicates reaction tubes with calcein color change.

Figure 4. Sensitivity test of the LAMP assay using DNA isolated from spiked urine samples by extraction and the direct boiling method for comparison. The LAMP reaction was performed at 65 °C for 30 min.

(A) Realtime turbidimetry result of the LAMP reaction. The black arrow on the graph of 1 × 10³ leptospires/ml indicates the shorter amplification time when tested on DNA extracted with a commercial extraction kit compared with DNA prepared by the direct lysis method. (B) (i) Colorimetric observation based on calcein dye color change. (i) Using DNA samples prepared by the direct lysis method (ii). Using DNA samples prepared by the column purification method. Tubes 1 to 6: 1 × 10⁵ to 1 leptospires/ml, Tube 7: non-spiked urine, PC, Positive control with secY plasmid; NC, no-template control. Red square box indicates reaction tubes with calcein color change.