RespiFinder 2SMART

Features and Benefits

• 18 viral + 4 bacterial RTI causing pathogens in one assay
• Diagnosis in 2.5 hours after nucleic acid extraction
• Detection based on melting curve analysis
• As sensitive as monoplex real-time PCR
• Internal Control included
• Amplification Controls included
• Positive Control included
• Interpretation software available

Overview

Acute respiratory tract infection (RTI) is the most widespread type of acute infection in adults and children and is a significant cause of disease in immunocompromised patients. Both viruses and bacteria can cause acute RTI, and the number of causative pathogens is large and diverse.

RespiFinder® 2SMART is a ready to use set of primers, enzymes and additional reagents for the simultaneous detection and differentiation of 22 respiratory pathogens, with the same sensitivity and specificity as monoplex Real Time PCR. Detection is based on melting curve analysis.

Sensitive and comprehensive diagnosis of the causative pathogen(s) can easily be achieved within a single working day. These fast resuts will improve therapy and will assist in the prevention of unnecessary use of antiobiotics or hospital admissions.

Targets

Detection systems

RespiFinder®  2SMART validated using the LightCycler® 480 of Roche and Rotor-Gene Q® of QIAGEN.

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Improvements

RespiFinder® 2SMART has been improved regarding  the previous RespiFinder product: RespiFinder® SMART 22 FAST v2.0 on:

• Faster time to result: results within 2.5 hours
• Simplified procedure: two steps-protocol
• Addition of Amplification Controls
• Addition of Positive Control
• Availability of interpretation software

Analysis and Interpretation

RespiFinder®  2SMART makes use of melting curve analysis for the detection of the pathogen(s) present in the sample.

The reverse primer of the signal amplification step contains a FAM fluorescent label. This FAM label is incorporated in the amplified sequences. Detection probes used for pathogen detection are labeled with ROX or CY5. After amplification a melt is performed. The melting curve analysis reveals melting peaks with a specific melting temperature in either FAM/ROX or FAM/Cy5 detection channel for each pathogen present in the sample. BHQ1 labels are used for detection of the Internal Control and Amplification Controls. Since BHQ1 is a quencer and not a fluorescent label, melting curve analysis of Internal Control and Amplification Controls reveal a negative melting peak in the FAM detection channel.

Procedure

After a gene-specific multiplex reverse transcription step, a signal amplification step is performed using a universal PCR primer pair of which one primer is labeled with a fluorescent dye (FAM).

The detection of the amplified FAM labelled probes is by melting curve analysis on a real-time PCR system. Twelve detection probes, either ROX or Cy5 labelled and varying in melting temperature, enable specific detection of the amplified product and the corresponding pathogen.

The Internal  Control (IC), which is added at the start of the procedure, and is detected by a specific detection probe in an additional channel, is included in the assay to validate a negative sample result.

Two Amplification Controls (AC) are provided in the signal amplification step to control for a correct amplification procedure.

Internal Amplification Control

The RespiFinder®  2SMART contains an Internal Control which is added to the sample in the nucleic acid extraction procedure. The Internal Control is supplied as a control for the RespiFinder®  2SMART procedure and to check for possible PCR inhibitors present in the sample.

A negative test result is validated by the presence of the Internal Control result.

Quality

• Designed and manufactured under EN ISO 13485:2012
• Validated on QCMD panels
• Validated on clinical samples

For Research Use Only.