Direct mecA Detection from Blood Culture Bottles by Branched-DNA Signal Amplification

Abstract

A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.

Rapid Detection of Methicillin-Resistant Staphylococci from Blood Culture Bottles by Using a Multiplex PCR Assay

“…We describe here a simple yet rapid method for detection of methicillin-resistant staphylococci from positive blood cultures. Although there have been several reports in the literature describing PCR-based methods for direct detection of resistant staphylococci (8,12,14,20,23), none have been optimized to allow both rapid DNA extraction and rapid PCR and detection. A potential limitation of this method is that the assay would not be able to distinguish the presence of MRSA in a blood culture from a culture that yielded a mixed growth of coagulase-negative and methicillin-susceptible staphylococci.…”

“…Several investigators have described PCR-based assays for detection of methicillin-resistant staphylococci from clinical samples (8,12,14,20,23). Some reports have shown that a pure culture of the organism is required (20), while other methods involving direct detection from clinical specimens require long DNA extraction protocols (14).…”

“…Jaffe et al reported a rapid method for extracting DNA from blood cultures, but it required several individual PCRs to detect all necessary products for identification of staphylococci (8). While claiming to be more rapid than conventional culture and susceptibility testing, some methods may still include many steps for DNA extraction and long PCR protocols requiring several hours (12,23). We describe a rapid and simple multiplex PCR-based method to detect methicillin-resistant staphylococci directly from positive blood culture bottles, dramatically reducing the time for identification from 24 to 48 h to approximately 3 h.…”

Genetic Methods for Assessing Antimicrobial Resistance

“…Signal generation is proportional to the amount of mecA gene sequences in the sample, which in turn is proportional to the number of staphylococcal cells analyzed. We have found the mecA bDNA assay to be as accurate as conventional PCR assays for detecting the mecA gene (29,84). The procedure is partly automated but requires approximately 6 to 8 h to complete.…”

“…It has been used to determine blood levels (viral loads) of HIV (25) and HCV (12). Our laboratory has adapted this assay to detect the mecA gene directly from blood cultures containing staphylococci (84). The bDNA method depends on the amplification of a signal molecule and not the target nucleic acid (Fig.…”