Legionella qPCR detection Kit

Legionella pneumophila is a pathogenic bacterium primarily known for causing Legionnaires’ disease, a severe form of pneumonia. A crucial genetic marker for detecting this bacterium is the macrophage infectivity potentiator (Mip) gene. The Mip gene encodes a peptidyl-prolyl cis/trans isomerase enzyme, essential for bacterial entry, intracellular replication, and survival within host macrophages. Due to its high conservation and specificity within Legionella species, the Mip gene region serves as a reliable molecular target for PCR-based diagnostics and environmental monitoring.

Attogene’s qPCR kit for Legionella is designed for the in vitro analysis of the crucial genetic marker Mip. A gene-specific primer mix targeting the Mip region is provided for amplification and detection using SYBR Green dye on a qPCR instrument. samples are collected and processed to extract purified genomic DNA (gDNA). A reaction mixture is prepared using provided primers, SYBR Green master mix, and extracted gDNA samples as required. The assembled reaction is then loaded onto the qPCR instrument for amplification. The primer mix utilizes Taq polymerase to amplify the gene region of interest, with the SYBR Green dye binding specifically to double-stranded DNA during PCR amplification. This allows for real-time fluorescence detection across a wide range of qPCR platforms.

Recommended DNA isolation technique – inquire at sales@attogene.com for further details:

1. Sample Collection and Filtration:

1. Collect water samples in sterile bottles, keeping them on ice or RT.
2. Assemble the filtration system with a 0.2 µm or 0.45 µm membrane filter in the plastic membrane housing.
3. Pass 100 mL to 1 L of water through the filter using a syringe.
4. Carefully remove the membrane using sterile forceps and transfer it to a sterile 2.0 mL microcentrifuge tube.
5. Store the membrane at -20°C (short-term) or -80°C (long-term) until DNA extraction.

2. Cell Lysis and DNA Extraction

1. Cut the membrane into small pieces with a sterile scalpel and transfer to a 1.5 mL microcentrifuge tube.
2. Add 500 µL of lysis buffer and 20 µL of Proteinase K.
3. Incubate at 60°C for 30–60 minutes with occasional vertexing.
4. Run through DNA extraction kit protocol or sonicate

3. Quantification and quality control

1. Measure DNA concentration using a spectrophotometer.
2. Use agarose gel electrophoresis to verify DNA integrity.
3. Perform PCR amplification of internal control genes to confirm successful extraction.