Evaluation of the analytical sensitivity and specificity of the test system for detecting the DNA of pathogenic leptospirae of the species Leptospira interrogans

Abstract

Leptospirosis belongs to socially significant natural focal infections. Laboratory diagnosis of leptospirosis includes bacteriological, serological, and molecular genetic studies. The analytical sensitivity and specificity of the diagnostic tool being developed is an important requirement for the creation of valid test systems which, in turn, will contribute to effective monitoring and control of the spread of leptospirosis in populations. This paper discusses the results of determining the analytical sensitivity and specificity of a PCR test system developed by the authors for detecting the DNA of pathogenic leptospira species Leptospira interrogans in biological samples and environmental objects. The developed PCR test system includes a combination of oligonucleotide primers flanking the DNA region of the lipL32 gene of L. interrogans. The analytical sensitivity of the test system was determined using 10-fold dilutions of samples contaminated with L. interrogans. It was found that the minimum amount of DNA in the sample of the test material was 200 copies per mL of the sample. To determine the analytical specificity, the samples were artificially contaminated with bacterial cultures of Brucella abortus, Escherichia coli, Mycobacterium bovis, and L. interrogans. The results of determining the analytical specificity of the test system showed that only samples with L. interrogans DNA underwent amplification. The samples with other bacterial contaminants gave a negative result. Thus, the analytical specificity of the developed test system for the indication of pathogenic leptospira by RT-PCR made 100%. The created test system for the diagnosis of pathogenic leptospirae by RT-PCR may be used in laboratory practice for the lifetime control of animals for leptospira transmission as well as for the indication of leptospirae in environmental objects.