Multiplex compositions and methods for quantification of human nuclear DNA and human male DNA and detection of PCR inhibitors
The invention relates to a method for simultaneous quantification of human nuclear DNA and human male DNA in a biological sample while also detecting the presence of PCR inhibitors in a single reaction. The multiplex quantification method also provides a ratio of human nuclear and male DNA present in a biological sample. Such sample characterization is useful for achieving efficient and accurate results in downstream molecular techniques such as genotyping.
1. A kit comprising primer pairs and an internal plasmid control, wherein at least one primer pair is capable of specifically amplifying human Ribonuclease P RNA component H1 (RPPH1), at least one primer pair is capable of specifically amplifying human sex determining region Y (SRY) loci and at least one primer pair is capable of specifically amplifying SEQ ID NO:1 and wherein the internal plasmid control comprises SEQ ID NO:1.
2. The kit of claim 1 , further comprising labeled probes for detecting the amplified human RPPH1 locus, the amplified human SRY locus, and amplified SEQ ID NO:1.
3. The kit of claim 2 , wherein the RPPH1 probe is labeled with FAM; the SRY probe is labeled with VIC, and the SEQ ID NO:1 probe is labeled with NED.
4. The kit of claim 1 , further comprising PCR reaction mix, a quantification standard, and a dilution buffer.
5. A kit comprising primer pairs and an internal plasmid control, wherein at least one primer pair is capable of specifically amplifying human RPPH1, at least one primer pair is capable of specifically amplifying human SRY loci and at least one primer pair is capable of specifically amplifying SEQ ID NO:2 and wherein the internal plasmid control comprises SEQ ID NO:2.
6. The kit of claim 5 , further comprising a labeled probe for detecting the amplified human RPPH1 locus, the amplified human SRY locus, and SEQ ID NO:2.
7. The kit of claim 6 wherein the RPPH1 probe is labeled with FAM; the SRY probe is labeled with VIC, and the SEQ ID NO:2 probe is labeled with NED.
8. The kit of claim 5 , further comprising PCR reaction mix, a quantification standard, and a dilution buffer.