Evaluation of the alkaline wash/lysis procedure for the molecular diagnosis of a positive bacterial blood culture in clinical routine practice

Abstract

Blood culture is commonly used to detect microorganisms in patients with a suspected blood infection. This study evaluated the alkaline wash/lysis procedure to extract DNA of microorganisms in a clinical blood culture. A multiplex polymerase chain reaction (PCR) targeting the 16S rDNA (ribosomal DNA) gene and the fungal ITS (internal transcribed spacer) gene was used as a reliable indicator for the presence of microorganism DNA in the extracts. A total of 535 BacT/ALERT positive blood culture bottles were evaluated. Multiplex PCR showed positive results in 530 DNA extracts, but 5 DNA extracts gave negative results. We conclude that the alkaline wash/lysis procedure in combination with the multiplex PCR is a simple and sensitive method, which can be used in a standard diagnostic laboratory to detect microorganisms in blood culture material.

Rapid Detection of bla KPC Carbapenemase Genes by Internally Controlled Real-Time PCR Assay Using Bactec Blood Culture Bottles

“…The assay, which includes human gene RNase P as an internal control, has been optimized to circumvent the inhibitors present in the broth of Bactec bottles and to be completed in less than 4 h. This assay also showed that once BSA fraction V (96%) was utilized as an inhibitor reliever, no manipulation of the samples was required before or after manual or automated extraction, as previously reported (10,14).…”