FIG. 1.
Comparative lower limit of detection of the tox real-time PCR assay and standard PCR. Corynebacterium diphtheriae (NCTC 10648) was grown on sheep blood agar at 37°C for 16 h, and serial 10-fold dilutions were prepared in HI broth. DNA was extracted from undiluted culture and from 10−1 to 10−8 dilutions and used in the tox real-time PCR assay and in the standard PCR assay. (A) Real-time PCR amplification generated by the Prism 7700 sequence detector showing amplification of target DNA from nine samples (from left to right: undiluted C. diphtheriae growth in HI broth and dilutions 10−1 to 10−8). ΔRn, difference in reporter fluorescence between the sample and the no-template controls; Ct, threshold cycle (i.e., the cycle at which a statistically significant increase in fluorescence is first detected). (B) Amplification of the A subunit of the tox gene (primers tox 1 and tox 2) by standard PCR. Lanes: L, low-mass DNA ladder; C, undiluted C. diphtheriae growth in HI broth; −, no-template control; 1 to 10, dilutions 10−1 to 10−10, respectively.