CMV PCR Kit

Description

The CMV PCR Kit constitutes a ready-to use system for the detection of CMV DNA using polymerase chain reaction (PCR) in the ABI PRISM® 7000, 7700 and 7900HT Sequence Detection System (Applied Biosystems).  The CMV Master contains reagents and enzymes for the specific amplification of a 105 bp region of the CMV genome.  The amplicon is detected by measuring the FAM fluorescence in the ABI PRISM® SDS.  In addition, the CMV PCR Kit contains a second heterologous amplification system to identify possible PCR inhibition.  This is detected as an Internal Control (IC) by measuring the VIC/JOE fluorescence.  The detection limit of the analytical CMV PCR is not reduced.  External positive controls (CMV QS 1-4) are supplied which allow the determination of the pathogen load.

Internal Control
An Internal Control (CMV IC) is supplied.  This allows the user both to control the DNA isolation procedure and to check for possible PCR inhibition.  For this application, add the Internal Control  to the isolation at a ratio of 0.1 µl per 1 µl elution volume.  For example, using the QIAamp DNA Mini Kit (QIAGEN) the DNA is eluted in 200 µl AE buffer.  Hence, 20 µl of the Internal Control should be added initially.  If you elute e.g. in 100 µl, then use the corresponding volume of 10 µl.  The quantity of Internal Control used depends only on the elution volume.  Please note that the Internal Control should be added to the mixture of lysis buffer and sample material.  Alternatively, the Internal Control can be added directly to the lysis buffer.  Optionally, you can add the carrier RNA together with the Internal Control to the lysis buffer.  However, please note that the mixture of Internal Control and lysis buffer/carrier RNA has to be prepared freshly and used instantly (storage of the mixture at room temperature or in the refrigerator for only a few hours may lead to Internal Control failure and a reduced extraction efficiency).  Please do not add the Internal control to the sample material directly!

The Internal Control can be optionally be used exclusively to check for possible PCR inhibition.  For this application, add 2 µl of the Internal Control and 5 µl CMV Mg-Sol per reaction directly to 25 µl CMV Master.  For each PCR reaction use 30 µl of the Master Mix produced as described above and add 20 µl of the purified sample.  If you are preparing a PCR run for several samples please increase the volume of the CMV Master, the CMV Mg-Sol and the Internal Control according to the number of samples.