MilliPROBE™ Real-Time Detection System for Pseudomonas aeruginosa

Description

MilliPROBE is an in-process early warning system that will provide you with faster, more effective detection of Pseudomonas aeruginosa. It is the first system that combines both speed and sensitivity in one screening tool enabling you to detect any potential P. aeruginosa contamination events earlier in your process. Real-time detection allows you the opportunity to perform any corrective action earlier in your production process significantly reducing downstream processing risks.

The MilliPROBE system for P. aeruginosa was designed to ensure a higher degree of water quality throughout your manufacturing process. The quality of your purified water system can significantly affect your operations and product quality. The MilliPROBE system provides presence/absence results within four hours, compared to days for traditional detection methods, enabling you to pinpoint contamination earlier, limiting the impact of the event and reducing downtime.

Benefits

   • Specific detection of P. aeruginosa
   • Reduces processing risk and increases operational efficiency
   • Optimizes product yield, reduces potential scrap and releases water and product holds faster
   • Streamlines your final documentation and approval process
   • Delivers sensitive, reliable results

About P. aeruginosa

P. aeruginosa is a common organism found in water and associated environments where nutrients are limited, such as in purified water systems or water for injection (WFI). Biofilms from P. aeruginosa can build up undetected for long periods of time in closed plumbing systems and equipment. Contamination events are often undetected until shedding of biomass occurs in your process, raw materials or final product formulations. This can result in significant quality issues, costly decontamination processes, and unanticipated down-time.

P. aeruginosa is considered an objectionable organism by regulatory agencies because the endotoxins it produces can cause serious disease in susceptible, immune compromised individuals. Its presence often indicates the presence of other waterborne microbes that can affect the quality of your final product.

Target applications

   • Purified water system and water for injection monitoring
   • Environmental monitoring
   • Clean-in-Place (CIP) verification
   • Biofilm detection and prevention
   • Raw material or final product monitoring*

An Innovative Solution for Microbial Detection

As a result of a collaboration between Millipore and Gen-Probe, the MilliPROBE system combines industry accepted, easy-to-use membrane filtration methodologies with advanced, proven nucleic acid technologies to deliver fast, sensitive and reliable microbial detection results
in one solution.

Sample preparation utilizes Millipore’s Milliflex® filtration system, which has been used throughout the Pharmaceutical Industry and complies with worldwide pharmacopeias (European, US, Japan). Unlike traditional detection methods, the MilliPROBE system is completely culture-free. Instead it utilizes proprietary Target Capture and Real-Time Transcription-Mediated Amplification (TMA) technologies** to concentrate, purify, and amplify ribosomal RNA (rRNA) for the detection of targeted microbial contamination. Multiple oligonucleotide sequence-specific steps are used to ensure that the intended specificity is achieved with every MilliPROBE test.

From sample prep to data analysis, the MilliPROBE system provides results within 4 hours yielding unmatched sensitivity for the detection of low levels of P. aeruginosa contamination earlier in your process.

** Target Capture and Real-Time Transcription-Mediated Amplification (TMA) are technologies of Gen-Probe Incorporated.

A Complete Integrated Solution

MilliPROBE sample processing consists of sample preparation, Target Capture, amplification and detection, and automated data analysis. Each step is integrated to ensure a seamless, efficient process. All reagents are manufactured and quality controlled to meet MilliPROBE assay performance specifications.

Sample Preparation

Samples are processed using standard membrane filtration. A Lysis Reagent is added to the membrane and briefly incubated to release and stabilize any microbial rRNA before it is drawn through the membrane and collected for further processing (Figure 1).

Figure 1. Sample Preparation: Filtration and Lysis

Advantages of Targeting rRNA

Each bacterial cell contains:

   • One to several copies of DNA
   • Up to 10,000 copies of ribosomal RNA

Specificity is achieved by targeting conserved or unique rRNA sequences.

Targeting ribosomal RNA

   • Higher sensitivity; fewer false negatives

Targeting ribosomal RNA

Target Capture

Using the Thermo Scientific KingFisher® mL instrument, the stabilized rRNA is purified by automated Target Capture. The lysate is transferred to a tube with Target Capture Reagent, which consists of magnetic beads, buffer and capture oligonucleotides, and heated. Target rRNA present is captured on the magnetic beads and cell debris, proteins, and non-specific DNA and RNA are washed away from the target (Figure 2). The rRNA on the beads is resuspended in Amplification Reagent.

Amplification and Detection

Target rRNA on the beads is resuspended in the Amplification Reagent and is then transferred to a microplate and placed in the PTI FluoDia™ T70 microplate reader. Target rRNA present is amplified and detected in real-time by hybridization of fluorescently labeled probes called molecular torches (Figure 3). The molecular torch probes are non-fluorescent unless bound to amplified target. As the rRNA is amplified, a fluorescent signal is generated. This signal is measured and recorded for the duration of the amplification reaction (Figure 4).

Figure 2. Target Capture Methodology

Figure 3. Real-Time Transcription-Mediated Amplification

Figure 4. Effectiveness of Real-Time Transcription-Mediated Amplification

Automated Data Analysis

The MilliPROBE data analysis software analyzes the raw data through the use of a proprietary algorithm and automatically generates a pass/fail result. The user-friendly, Windows®-based software (Figure 5) with 21 CFR Part 11 compliance-ready features allows data to be saved and displayed in both graphical and text reports. Convenient informational data storage fields allow easy access for tracking, trending and audit trails.

Figure 5. Automated Data Analysis

MilliPROBE Real-Time TMA FACTS

   • No growth or pre-enrichment step required
   • Targets rRNA
   • Utilizes a single primer and two enzymes

   •    Reverse transcriptase
   •    T7 RNA polymerase

   • Amplifies RNA to RNA via DNA intermediates
   • Isothermal reaction (no thermal-cycler required)

Specifications (return to top)