Evaluation of three qPCR for the detection of pathogenic leptospires in domestic animals in Nicaragua.

Introduction

Molecular biology diagnostic methods such as real-time PCR should be used in Nicaragua to improve the diagnosis of leptospirosis in humans and animals.

Objective

To evaluate three qPCR methods for pathogenic Leptospira detection in domestic animals. Materials and methods: Real-time PCR primers were designed for the amplification of specific regions from the Lip 32 gene of Leptospira in SYBER Green (SYBER Green-A) and TaqMan, as well in SYBER Green-B as previously published. The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to select probes and primers. The analytical sensitivity was determined by calculating the detectable genomic equivalent while 18 pathogenic references strains and 28 negative controls were used to evaluate the sensitivity and specificity of each one of the three sets in 129 urine samples of domestic animals.

Results

The detection limit of four genomic equivalents per reaction was obtained from SYBR Green-A. The specificities were 94.4% (95% CI: 81.1-100.0) for TaqMan, 77.8% (95% CI: 55.8-99.8) for SYBR Green-A, while for SYBR Green-B it was 61.1% (95% CI: 35.8-86.4). In the three tests, we obtained a specificity of 100% (95% CI: 98.2-100.0). In the field samples, 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B.

Conclusion

SYBR Green-A presented the lowest detection limit while the three techniques under evaluation showed high specificity while TaqMan was the most sensitive.