Human health depends on the ability to detect extremely low levels of pathogenic bacteria. Early diagnosis of human infection is needed for effective patient treatment, and delayed diagnosis can lead to serious health problems or even death. In food, trace levels of bacteria can multiply to dangerous levels during transit, display, and storage, even at refrigerated temperatures. Detecting trace levels of bacteria in environmental or air samples enables identification and accurate geographical delineation of a potential bioagent release in the event of a terrorist attack or during military operations. In all these cases, speed is of the essence, yet standard methods to identify bacteria require 24 to 48 hours using traditional enrichment media and selective culturing on agar plates or identification via molecular methods. These methods are time-consuming, require manual manipulation, and cannot detect trace levels of bacteria.

Pacific Northwest National Laboratory’s rapid detection process for microorganisms rapidly enriches and detects even trace levels of microorganisms with minimal manual input. The process couples polymerase chain reaction (PCR)—a well-accepted and highly sensitive laboratory procedure—with gene-matched enrichment media. This enrichment media is built from a modified microorganism containing a variety of mutations in a selected gene. These modified microorganisms do not contain the signature genes a PCR assay would recognize. In a sense, the media “tricks” the PCR instrument into sensing a larger or amplified sample, allowing the identification of the microorganism of interest.

The technology consists of a process to create gene-matched enrichment media, kits containing such media for certain microorganisms, isolated nucleic acid molecules to prime the process, and a procedure to detect microorganisms using PCR. The process, kits, primers, and procedure can be used to detect the presence of any one of a number of different microorganisms, including pathogenic microorganisms. Samples can be obtained from a subject or collected from food or the environment.

While many traditional approaches cannot determine the presence of microbes at less than 1,000 CFU per milliliter of sample (where CFU stands for colony-forming unit, a measure of microbial viability), PNNL’s approach can determine as little as 10 CFU, allowing identification of pathogenic microbes faster and more accurately than any other approach on the market. This speed and accuracy is particularly important when tracking the emergence of virulent bacterial diseases or the spread of biohazards from a terrorist attack.

Applicability

Rapid detection of pathogenic organisms is important in many applications, including food safety, clinical diagnostics, homeland security, research, and defense. PNNL’s rapid detection process can be used to detect any type of microorganism, including bacteria, fungi, protozoa, and nematodes. The technology is particularly suited to applications that require the detection of very low levels of a microorganism in a given sample, and for detection of otherwise slow-growing microorganisms.