Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) -based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207) blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira) culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21%) out of 100 culture positive blood samples, three (2.8%) out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001). A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001) signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

Construction of the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans and the immuno-reactivity of its expressed products / 中国人兽共患病学报

In the present study, the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans was constructed by using PCR with linking primers, expressed in prokaryotic expression system. And the immuno-reactivity of its expressed product was determined. Meanwhile, SDS-PAGE and BioRad agarose image analyzer system was used to examine the expression output of the target recombinant protein rLipL32/1-LipL41/2, and the immune-reactivity of this recombinant protein was identified by Western blot assay. ELISA was used to detect the level of antibodies against the recombinant proteins in sera of patients with leptospirosis. It was demonstrated that the percentages of similarity in nucleotide and the putative amino acid sequences of the fusion gene lipL32/1-lipL41/2 with the corresponding sequences previously reported were 99. 9% and 98. 9%respectively. The expression output of the target recombinant protein was approximately 10% of the total bacterial proteins.Both the rabbit antisera against rLipL32/1 and rLip41/2 could recognize and combine to the recombinant fusion protein rLip32/1-Lip41/2 as well. The positive rates of antibodies in 228 leptospirosis patients with the recombinant proteins rLip32/1,rLip41/2 and rLip32/1-Lip41.2 were 97.4%, 78. 5% and 99. 1% respectively. The results of the present study leads to the conclusion that the fusion gene lipL32/1-lipL41/3 with its prokaryotic expression system is successively constructed and the expressed recombinant protein shows excellent immuno-reactivity, thus suggesting that it may be used as the antigenic candidate for the development of the leptospiral genus-specific engineering vaccine and for the preparation of diagnostic kits.