Molecular analysis of cross-bacterial contamination detected in biotin-free buffers during diagnosis of HCV infections

Abstract

In a routine work to determine Hepatitis C Virus (HCV) molecules in human infected serum with biotin/streptavidine enzyme linked immunosorbant assay (ELISA) technique, unexpected false positive was observed. No positive signals were noticed after changing all ELISA buffers. Subsequently, contaminated buffers were screened and analyzed for microbial contamination. Out of fifty-five, five randomly selected bacterial colonies were examined for biosynthesis of biotin using ELISA and/or Western blot binding biotin techniques in both supernatants and cell pellets. In compare to the E.coli reference strain a strong biotin signals in all examined isolates were recorded. All isolates were then examined for their genetic heterogeneity by PCR-RFLP technique of 23S rDNA genes. While, isolate BP(R2) which gave the highest biotin signal, was subjected for molecular identification. Comparative sequence analysis of the 16S rDNA gene (~1440 bp) revealed that this isolate is a member of the bacterial genus Delftia exhibiting a similarity value of 99.3% with Delftia acidovorans. In conclusion, a soluble biotin is secreted by the isolate Delftia acidovorans BP(R2) and it is also coupled to protein with molecular weight 25-26 KDa. As well as, this bacterial contamination was the reason for the false positive results observed during the detection of HCV infections.


Journal of Applied Sciences and Environmental Management Vol. 9(1) 2005: 5-10