Evaluating an MRNA based body fluid identification test using sybr green fluorescent dye and real-time PCR

The requirement to have more definitive and wider ranging body fluid identification (BFI) tests has resulted in a range of mRNA based real-time PCR BFI assays utilising Taqman fluorescent dye. An attempt to make a reliable BFI test utilising the alternative SYBR Green fluorescent dye was carried out.

Samples were extracted from blood and saliva stains and then reverse transcribed using M-MLV and random hexamers. Using real-time PCR, relative quantitation of SPTB (blood), NCF2 (blood), KRT4 (saliva) and SPRR1A (saliva) was carried out on cDNA from the blood and saliva samples using the SYBR Green reagents. Melting curve analysis was also conducted following the amplification step. The RQ values were calculated using the formula 2-ΔΔCT.

In all cases, the blood markers (SPTB and NCF2) were under expressed in saliva and over expressed in blood. The saliva markers (KRT4 and SPRR1A) were over expressed in saliva and under expressed in blood. Verification of the amplicons was carried out using melting curve analysis.

Relatively high levels of fluorescence were also detected in the reverse transcription blanks and negative controls; despite stringent anti-contamination procedures. This along with the melting curve analysis results suggest that the amplification may be a by-product of the test itself rather than contamination; due to the lack of specificity of the SYBR Green fluorescent dye. This was verified by contamination monitoring using negative controls along with SYBR Green and Taqman fluorescent dyes.

This shows that a SYBR Green based BFI test could be developed and it may be more appropriate to use than a Taqman based BFI test in a commercial forensic science environment. However, the amplification detected in the reverse transcription blanks and negative controls may render the results of a SYBR Green based BFI test unreliable and insufficiently robust for use in a court of law.