Real-time PCR method for the detection of the gene encoding surface lipoprotein LipL32 of pathogenic Leptospira: use in the laboratory diagnosis of the acute form of leptospirosis
Abstract
Background: The aims of this work were to replace the obsolete PCR method for the laboratory diagnosis of the acute form of leptospirosis using the G1, G2 an B64 I, B64 II primers, and to improve the PCR detection time.Methods: We introduced a real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 of pathogenic Leptospira into our laboratory diagnosis of the acute fom of leptospirosis. The positive and negative analytical specificities of the real-time PCR method were both equal to 100%; the detection limit was determined to be 1-5 genome copies/1 ml of liquid biological material.The method was further validated on 230 laboratory strains of leptospires.
Results: All laboratory strains of pathogenic Leptospira were evaluated as LipL32-positive and all non-pathogenic strains as LipL32-negative. In addition, 455 biological materials (253 plasma, 121 urine, 72 cerebrospinal fluid (CSF), 7 bronchoalveolar lavage, and 2 sputum) from 295 patients with suspected leptospirosis were examined.
From this set of patients, 9 were evaluated to be LipL32-positive, from 15 positive biological material (10 urine, 4 blood plasma, and 1 CSF). Conclusions: This real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 is a reliable, sensitive, and rapid method for the detection of the acute form of leptospirosis.