A Loop-Mediated Isothermal Amplification Assay Utilizing Hydroxy Naphthol Blue (LAMP-HNB) for the Detection of Treponema pallidum Subspp. pallidum

Abstract

Treponema pallidum subspp. pallidum is a spirochaete bacterium that causes syphilis, one of the most common sexually transmitted diseases. Syphilis progresses through four distinct stages, each characterized by specific symptoms, namely primary, secondary, latent, and late (tertiary) syphilis. Serology has been considered the primary diagnostic approach. However, it is plagued by problems such as the limited specificity of nontreponemal tests and the inadequate correlation of treponemal tests with disease activity. In this study, we focused on the development of a loop-mediated isothermal amplification assay utilizing hydroxy naphthol blue (LAMP-HNB) for the diagnosis of T. pallidum subspp. pallidum. Specifically, this study seeks to determine the analytical sensitivity (limit of detection; LOD) and analytical specificity. Four hundred clinical serum samples were analyzed for diagnostic sensitivity, specificity, and predictive value, and each technique's 95% confidence intervals (95% CI, p < 0.05) were evaluated. The limit of detection for polymerase chain reaction with agarose gel electrophoresis (PCR-AGE), the loop-mediated isothermal amplification assay combined with agarose gel electrophoresis (LAMP-AGE), and LAMP-HNB were 116 pg/µL, 11.6 pg/µL, and 11.6 pg/ µL, respectively. Analytical specificity examinations indicated the absence of cross-reactivity with Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy human serum in PCR-AGE, LAMP-AGE, and LAMP-HNB. The diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV), and negative predictive value (NPV) for PCR-AGE were 100.00 (100.00)%, 94.50 (94.40-94.60)%, 94.79 (94.69-94.88)%, and 100.00 (100.00)%, respectively. While, for LAMP-AGE and LAMP-HNB, they were 100.00 (100.00)%, 91.00 (90.87-91.13)%, 91.74 (91.63-91.86)%, and 100.00 (100.00)%, respectively. The LAMP-HNB test is simple, rapid, highly sensitive, and highly specific, without requiring expensive equipment. In the future, the LAMP-HNB assay may develop into a single-step diagnostic process, enabling the use as point-of-care testing for the diagnosis, prevention, and management of syphilis infection.

Keywords: Treponema pallidum; hydroxy naphthol blue (HNB); loop-mediated isothermal amplification (LAMP).

Figure 1

The change in the color of hydroxy naphthol blue (HNB) is attributed to the decrease in magnesium. The concentration of Mg²⁺ in the solution decreases during the LAMP process, the color of the HNB solution changes from purple to blue, which can be observed with the naked eye after amplification.

Figure 2

The optimization of the LAMP assay in the range of 4.5–5.5 mM. Lane “M” represents a 100 bp plus DNA ladder marker of Vivantis, Darul Ehsan, Malaysia, and “N” is the negative control.

Figure 3

The optimization of thermal conditions ranging between 60 and 65 °C. Lane “M” represents a 100 bp plus DNA ladder marker of Vivantis, Darul Ehsan, Malaysia, and “Neg” is the negative control.

Figure 4

The optimal reaction durations were observed at 45 and 60 min. Lane “M” represents a 100 bp plus DNA ladder marker of Vivantis, Darul Ehsan, Malaysia, and “Neg” is the negative control.

Figure 5

The optimization of LAMP-HNB, ranging from 1 to 20 µM.

Figure 6

The analytical sensitivity and specificity tests and DNA analysis using 10-fold dilution. In the agarose gel electrophoresis (AGE) results, lanes 1–9: 11.6 ng/µL, 1.16 ng/µL, 116 pg/µL, 11.6 pg/µL, 1.16 pg/µL, 116 fg/µL, 11.6 fg/µL, 1.16 fg/µL, and negative control, respectively. Lane “M” represent a 100 bp plus DNA ladder marker of Vivantis. (a) PCR with agarose gel electrophoresis (PCR-AGE). (b) LAMP with agarose gel electrophoresis (LAMP-AGE). (c) LAMP utilizing hydroxy naphthol blue (LAMP-HNB).

Figure 7

In the agarose gel electrophoresis (AGE) results, lanes 1–11: T. pallidum subspp. pallidum, Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Human Immunodeficiency Virus (HIV), healthy human serum, and negative control, respectively. Lane “M” represent a 100 bp plus DNA ladder marker of Vivantis. (a) PCR with agarose gel electrophoresis (PCR-AGE). (b) LAMP with agarose gel electrophoresis (LAMP-AGE). (c) LAMP utilizing hydroxy naphthol blue (LAMP-HNB).