HSV-1/2 PCR Kit

Description

The HSV-1/2 PCR Kit constitutes a ready-to-use system for the detection and differentiation of herpes simplex virus-1 and -2DNA using polymerase chain reaction (PCR) in the ABI Prism 7000 and 7900HT Sequence Detection System (Applied Biosystems).  The HSV Master contains reagents and enzymens for the specific amplification of a 148 bp region of the herpes simplex virus genome.  The amplicon is detected by measuring the FAM fluorescence (HSV1) and NED fluorescence (HSV2) in the ABI PRISM SDS.  In addition, the HSV-1/2 PCR Kit contains a second heterologus amplification system to identify possible PCR inhibition.  This is detected as an Internal Control (IC) by measuring the VIC fluorescence.  The detection limit of the analytical HSV PCR is not reduced.  External positive controls (HSV1 QS 1-4 & HSV2 QS 1-4) are supplied which allow the determination of the pathogen load.

Internal Control
An Internal Control (HSV 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 0.1µl 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.  The Internal Control and carrier RNA should be added only

• to the mixture of lysis buffer and sample material or
• directly to the lysis buffer.

The Internal Control must not be added to the sample material directly.  If added to the lysis buffer 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 fridge for only a few hours may lead to Internal Control failure and a reduced extraction efficiency).  Please do not add the Internal Control and the carrier RNA to the sample material directly.

The Internal Control can optionally be used exclusively to check for possible PCR inhibition.  For this application, add 2 µl of the Internal Control and 10 µl HSV Mg-Sol per reaction directly to 20 µl HSV 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 HSV Master, the HSV Mg-Sol and the Internal Control according to the number of samples.