Real-time Pcr Assay for Detection of Tetracycline Resistance Genes of Gram-negative Bacteria

Doxycycline, a new generation tetracycline antibiotic, is currently one of choice for the treatment and prevention of infections caused by agents of biowarfare. We are developing real time PCR assays to detect tetracycline resistance genes in Gram-negative bacteria. The assay was developed as a multiplex SYBR Green I detection using the Roche Lightcycler and multi-melting peak analysis followed by a specific 5’ nuclease assay. Specific primer pairs were selected for the PCR amplification of seven tetracycline resistance genes commonly found in Gram-negative organisms. A combination of primer pairs were used in a multiplex PCR reaction with SYBR Green I to detect a group of Tet resistance genes: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G) and tet(H). Based on the melting peak analysis of our SYBR Green I reaction, we could preliminarily determine the class of the Tet resistance determinant that gave the positive signal. To confirm the result, we designed specific TaqMan primers and probes for each class of Tet determinant. Positive results were determined by comparing the Ct (crossing point) as well as DNA sequencing. We analyzed forty-eight clinical isolates by both assays. In 37 samples, the 5’ nuclease assay confirmed the identity shown in SYBR Green I multiplex PCR reaction. 4 samples confirmed to be one uncommon tet gene (Tet J resistance determinant). Other 6 keep unknown. Our results demonstrate that the multiplex PCR assay with SYBR Green I is a method of significant saving in terms of labor and time in strains analysis. The SYBR Green I assay coupled with a class-specific 5’ nuclease assay is a two-fold confirmation and identification of the Tet resistance genes present in Gram-negative organisms.