Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of Treponema pallidum DNA in the Peripheral Blood of Secondary Syphilis Patients

Abstract

Secondary syphilis (SS) has always been puzzling for the clinicians because of the similarity of the appearance of skin rashes with other dermatoses. Serological assays are useful, but less sensitive at an early stage of SS or when patients are immunodeficient. Therefore, there is an urgent need to develop a rapid and effective tool for the diagnosis of SS, which may play an important role in the control of epidemic syphilis outbreaks. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay, targeting gene encoding the basic membrane protein of Treponema pallidum, to detect the presence of circulating T. pallidum DNA in the blood of SS patients. The specificity of LAMP was validated using three strains of Spirochaetales and six common clinical bacteria. The clinical applicability of LAMP assay was assessed using 642 blood samples from clinically suspected SS patients and 80 samples from healthy blood donors, showing a sensitivity of 82.1% and a specificity of 100.0% in the diagnosis of SS. Thus, our results indicate that the LAMP can be used as a supplementary method for the diagnosis of SS.

Figure 1.

Agarose gel electrophoretic and fluorescence analysis of the loop-mediated isothermal amplification products at different temperature and time. (A and C) 57°C (lane 2), 59°C (lane 3), 61°C (lane 4), 63°C (lane 5), 65°C (lane 6), 67°C (lane 7) and 69°C (lane 8); negative control (lane 1). (B and D): 15 minutes (lane 2), 30 minutes (lane 3), 45 minutes (lane 4), 60 minutes (lane 5), 75 minutes (lane 6) and 90 minutes (lane 7); negative control (lane 1). The DNA size markers are indicated on the sides. This figure appears in color at www.ajtmh.org.

Figure 2.

The specificity of loop-mediated isothermal amplification (LAMP) and simple polymerase chain reaction (PCR). (A) Agarose gel electrophoretic analysis. (B) The fluorescence of LAMP reaction. (C) Agarose gel electrophoretic analysis of simple PCR of various bacteria strains. Lane 1 = Borrelia burgdorferi; lane 2 = Leptospira interrogans; lane 3 = Treponema denticola; lane 4 = Staphylococcus aureus; lane 5 = Enterococcus faecalis; lane 6 = Escherichia coli; lane 7 = Klebsiella pneumoniae; lane 8 = Acinetobacter baumannii; lane 9 = Pseudomonas aeruginosa; N = negative control (deionized water); P = positive control (T. pallidum Nichols strain); M = 100-bp DNA ladder. This figure appears in color at www.ajtmh.org.

Figure 3.

Agarose gels showing the detection sensitivity of loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) for detecting Treponema pallidum Nichols DNA. (A) 2.0% agarose gel of LAMP products. (B) 2.0% agarose gel of simple PCR amplification products. Lane 1 = 5.4 × 10⁻¹ ng/μL; lane 2 = 5.4 × 10⁻² ng/μL; lane 3 = 5.4 × 10⁻³ ng/μL; lane 4 = 5.4 × 10⁻⁴ ng/μL; lane 5 = 5.4 × 10⁻⁵ ng/μL; lane 6 = 5.4 × 10⁻⁶ ng/μL and lane 7 = 5.4 × 10⁻⁷ ng/μL; lane 8 = negative control.