Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction.

Abstract

The aim of this study was to develop polymerase spiral reaction (PSR) for rapid, sensitive and specific detection of Brucella sp. Polymerase spiral reaction assay was developed using specifically designed primers targeting the conserved multicopy IS711 gene of Brucella sp. The assay could be performed within 60min at an isothermal temperature of 64°C. The lower limit of detection of PSR was 11·8fg and conventional PCR was 1·18pg of Brucella abortus genomic DNA. Thus, PSR was found to be 100-fold more sensitive than conventional PCR and was comparable to real-time PCR. The specificity of PSR was tested with other non-Brucella bacteria and also with some bacterial and viral pathogens causing abortions. The assay was found to be specific as it did not detect any putative pathogens other than Brucella sp. Fifty-six clinical samples suspected for brucellosis (aborted fetal stomach content) were screened with PSR to validate the applicability of the test to detect Brucella DNA. The same samples were also screened with conventional PCR and real-time PCR. Of 56 samples, 25 samples were found to be positive with both PSR as well as real-time PCR, whereas only 20 samples were found positive with conventional PCR. The results of this study indicated that the PSR assay is a simple, rapid, sensitive and specific method for the detection of Brucella sp. that may improve diagnostic potential in clinical laboratories or can be used at diagnostic laboratories with minimal infrastructure. The PSR assay, because of its simplicity and low cost, can be preferred to other molecular methods in the diagnosis of infectious diseases.