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Using the new Phadebas® Forensic Press test to find crime scene saliva stains suitable for DNA analysis
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Forensic Science International: Genetics Supplement Series 1 (2008) 430–432 www.elsevier.com/locate/FSIGSS
Research article
Using the new Phadebas1 Forensic Press test to find crime scene saliva stains suitable for DNA analysis Johannes Hedman, Karin Gustavsson, Ricky Ansell * Swedish National Laboratory of Forensic Science, SKL, SE-581 94 Linko¨ping, Sweden Received 20 August 2007; accepted 1 October 2007
Abstract The Phadebas1 Forensic Press test is a new product that detects saliva stains by reacting with amylase. When the paper is pressed against a saliva stain a blue spot occurs. To test the sensitivity of the paper, a set of dilution series of saliva down to 1:500 was prepared on cotton fabric. Blue spots could be seen for dilutions of 1:100 when incubated at room temperature, and 1:200 in 37 8C. However, incubation at 37 8C did not provide acceptable reproducibility. The Phadebas1 test was compared to four different lightsources for the ability to detect saliva on different carrier materials. Cotton fabric (T-shirt), denim, suede, leather, painted wood and untreated wood were tested. On denim, no stains could be detected with the lightsources, but Phadebas1 showed all stains for pure saliva and dilution 1:5. DNA analysis was performed on detected stains and corresponding spots on the Phadebas1 paper. Complete DNA profiles were produced for stains from pure saliva and dilution 1:5. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Phadebas; Lightsource; Saliva stains; Stain detection; DNA analysis
1. Introduction Saliva stains with epithelial cells can be very useful in forensic DNA casework. At the Swedish National Laboratory of Forensic Science, non-specific lightsources are used to find saliva on different materials. A significant amount of retrieved samples, thought to contain epithelial cells, turn out to be blanks. Phadebas1 is a chemistry for saliva detection based on amylase activity and a water insoluble starch complex with a colour molecule attached. When the amylase breaks the complex, the colour is released. The system has been used in forensic casework for over three decades, involving rather laborious manual steps [1,2]. In early 2007, the new Phadebas1 Forensic Press test became commercially available. A filter paper has been pre-treated with the Phadebas1 chemistry. One side is blue and one white. When moistened and pressed against a positive saliva stain, with the white side facing upwards, a blue spot becomes visible. The objectives were to determine the sensitivity of the Phadebas1 Forensic Press test and the
* Corresponding author. Tel.: +46 13241435; fax: +46 13145715. E-mail address: [email protected] (R. Ansell). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.205
correlation between a positive Phadebas1 result and the possibility to obtain a DNA profile. 2. Material and methods The experiments were based on protocols available from the manufacturer [3]. The amylase levels in saliva vary between individuals, and for one person over time. Therefore saliva from two persons was collected on the day of each experiment, and analysed in parallel. To avoid cross contamination, the sampled persons did not take part in the laboratory work. One saliva dilution series (1:1, 1:5, 1:10, 1:25, 1:50, 1:100, 1:200, 1:500) was prepared for each sampled person, using physiological saline-solution as agent. A triplicate of 50 ml of each dilution was dispensed on cotton fabric (T-shirt), together with one negative control (50 ml of physiological salinesolution). For each dilution series, two identical pieces of cotton fabric were prepared for parallel development in room temperature and 37 8C (heating cabinet). The stains were left to dry in room temperature for approximately 2 h before analysis. The pieces of cotton fabric were put on a clean operation cloth and moistened using de-ionised water. A piece of Phadebas1 Forensic Press test paper was put on top of the fabric and moistened to saturate the paper, thereby starting the reaction. A glass plate was put on top of the paper and a weight used to
J. Hedman et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 430–432
press the paper tightly against the fabric. The progress of the reactions was monitored every 5 min. After 40 min the reaction was interrupted, and the paper left to dry. Six different carrier materials were studied: denim, cotton fabric (T-shirt), leather, suede, untreated wood and painted wood. Two saliva dilution series were prepared (1:1, 1:5, 1:25, 1:100). Triplicates of 50 ml of each dilution were dispensed on each set of materials, together with one negative control. The exhibits were left to dry over night, and studied using four different lightsources: Crime Scope1 CS-16-10, 430 nm/ yellow goggles (Spex); Quaser 2000/30, 340-413 nm/transparent goggles (Foster & Freeman); UV spotlight, 365 nm/ transparent goggles (Labino) and Polilight1 PL6, 425 nm/ yellow goggles (Rofin). Phadebas1 development was performed approximately 24 h after application of the stains, following the protocol described above (room temperature). After drying the exhibits and the Phadebas1 paper, sampling for DNA analysis was performed. Two stains, of two chosen saliva concentrations (1:1 and the lowest concentration showing a positive Phadebas1 result), from each exhibit and the corresponding Phadebas1 paper were sampled. The stains on untreated wood, painted wood, leather and suede were swabbed using cotton swabs, cut and placed in microfuge tubes. The stains on cotton fabric, denim and the Phadebas1 papers were cut directly (1.5 cm 1.5 cm) into microfuge tubes. Chelex 1001 extraction was used for all samples [4]. For leather and suede a step of Centricon1-100 tube purification was performed [5]. The DNA analysis was performed using Applied Biosystems 7300 Real Time PCR System with QuantifilerTM Human DNA Quantification Kit, GeneAmp1 PCR System 9700 with AmpFISTR1 SGM Plus1 PCR Amplification Kit, ABI PRISM1 3130xl Genetic Analyzer and GeneMapperTM ID v3.1. 3. Results and discussion With pure saliva, strong colour changes could be seen for all stains within 5 min. After 40 min, stains were visible down to dilution 1:25 for both room temperature series and one 37 8C series. Weak results were observed for dilution 1:100 at room temperature and for 1:200 at 37 8C (one series). The second 37 8C series only gave results to 1:10. After drying of the Phadebas1 paper, strong results could be seen down to dilution 1:50 at room temperature. For all positive results, blue colour from the paper was displaced onto the cotton fabric, generating a visible detection on the examined exhibits. No results were seen for the negative controls. The inconsistency for incubation in 37 8C is probably caused by elevated evaporation, and the problem to moisten the material and paper evenly and enough. Since room temperature showed more even results, and is handier to apply both at crime scenes and in the laboratory, this was chosen for the remaining experiments. However, the indications of lower detection levels at higher temperatures should be evaluated further. On denim, no stains could be detected using the lightsources, not even for pure saliva. Using Phadebas1, both the 1:1 and 1:5 dilutions were clearly visible after 40 min of incubation. On
431
cotton fabric, Phadebas1 gave results down to 1:100, whereas the lightsources failed for pure saliva. However, all lightsources except Crime Scope1 visualised stains for the saline negative controls, indicating that something else than saliva was fluorescing. For untreated wood, the UV spotlight gave fluorescence for all stains including the negative control, whereas the other lightsources failed to visualise any stains. Phadebas1 showed clear stains down to dilution 1:100. On leather, Phadebas1 and the UV spotlight gave results down to 1:25, the Quaser only managed concentrated saliva. The other lightsources failed to visualise any stains. For painted wood, the lightsources visualised stains down to 1:25, whereas Phadebas1 clearly showed all stains down to 1:100. On suede, Phadebas1 showed no stains, whereas Crime Scope1 and UV spotlight gave results down to 1:25 and 1:100, respectively. However, Crime Scope1 visualised stains for the saline negative control as well. The Phadebas1 test generated better results than any lightsource for all materials, except for suede. The comparative advantage of the saliva specific amylase method versus the lightsources is obvious. However, the longer detection time and invasive character of the method might benefit use of lightsources. It is clear that highly absorbing surfaces, such as suede and denim, are troublesome for both amylase based and lightsource detection, as the saliva appears trapped inside the texture of these exhibits. Still, Phadebas1 seemed to work better on these material types. It is clear that it is very important to moisturise the Phadebas1 paper and the exhibit well, especially for highly absorbing materials. Some problems during this project emanated from not using enough water and this has to be considered in further studies. The DNA analyses of pure saliva stains on different materials generated complete profiles in all cases tested. For leather and painted wood, the DNA analyses of the corresponding piece of Phadebas1 paper also yielded complete profiles, and the amount of DNA was approximately the same as from the exhibits. For the other exhibits, the paper samples failed to produce any analysable amounts of DNA. The plausible reason for this is that less absorbent or inert exhibits are more prone to transfer cells to the Phadebas1 paper. Of the samples from the lowest detected stains, only the 1:5 dilutions (denim and leather) gave enough DNA to enable analysis. This suggests that the Phadebas1 test is more sensitive for saliva stain detection than the standard methods for forensic DNA analysis. The 1:100 dilutions that were successfully detected on some materials correspond to 0.5 ml pure saliva in the 50 ml stain, which is a very minute amount when it comes to DNA analysis. The detection levels for Phadebas1 are also highly dependent on the nature of the background material. No PCR inhibition could be seen in the analysis, suggesting that the Phadebas1 chemistry does not interfere with the subsequent PCR. In conclusion, the Phadebas1 Forensic Press test gives a good indication to whether or not a crime scene stain contains saliva suitable for DNA analysis. However, positive stains might contain too little DNA and negative stains might produce complete DNA profiles.
432
J. Hedman et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 430–432
Conflict of interest None. References [1] G.M. Willot, An improved test for the detection of salivary amylase in stains, J. Forensic Sci. Soc. 14 (1974) 341–344.
[2] G.M. Willott, M. Griffiths, A new method for locating saliva stains—Spotty paper for spotting spit, Forensic Sci. Int. 15 (1980) 79–83. [3] Magle Life Sciences. http://www.phadebas.com. [4] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 1001 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (4) (1991) 506–513. [5] Millipore. http://www.millipore.com.
Forensic Science International: Genetics Supplement Series 1 (2008) 430–432 www.elsevier.com/locate/FSIGSS
Research article
Using the new Phadebas1 Forensic Press test to find crime scene saliva stains suitable for DNA analysis Johannes Hedman, Karin Gustavsson, Ricky Ansell * Swedish National Laboratory of Forensic Science, SKL, SE-581 94 Linko¨ping, Sweden Received 20 August 2007; accepted 1 October 2007
Abstract The Phadebas1 Forensic Press test is a new product that detects saliva stains by reacting with amylase. When the paper is pressed against a saliva stain a blue spot occurs. To test the sensitivity of the paper, a set of dilution series of saliva down to 1:500 was prepared on cotton fabric. Blue spots could be seen for dilutions of 1:100 when incubated at room temperature, and 1:200 in 37 8C. However, incubation at 37 8C did not provide acceptable reproducibility. The Phadebas1 test was compared to four different lightsources for the ability to detect saliva on different carrier materials. Cotton fabric (T-shirt), denim, suede, leather, painted wood and untreated wood were tested. On denim, no stains could be detected with the lightsources, but Phadebas1 showed all stains for pure saliva and dilution 1:5. DNA analysis was performed on detected stains and corresponding spots on the Phadebas1 paper. Complete DNA profiles were produced for stains from pure saliva and dilution 1:5. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Phadebas; Lightsource; Saliva stains; Stain detection; DNA analysis
1. Introduction Saliva stains with epithelial cells can be very useful in forensic DNA casework. At the Swedish National Laboratory of Forensic Science, non-specific lightsources are used to find saliva on different materials. A significant amount of retrieved samples, thought to contain epithelial cells, turn out to be blanks. Phadebas1 is a chemistry for saliva detection based on amylase activity and a water insoluble starch complex with a colour molecule attached. When the amylase breaks the complex, the colour is released. The system has been used in forensic casework for over three decades, involving rather laborious manual steps [1,2]. In early 2007, the new Phadebas1 Forensic Press test became commercially available. A filter paper has been pre-treated with the Phadebas1 chemistry. One side is blue and one white. When moistened and pressed against a positive saliva stain, with the white side facing upwards, a blue spot becomes visible. The objectives were to determine the sensitivity of the Phadebas1 Forensic Press test and the
* Corresponding author. Tel.: +46 13241435; fax: +46 13145715. E-mail address: [email protected] (R. Ansell). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.205
correlation between a positive Phadebas1 result and the possibility to obtain a DNA profile. 2. Material and methods The experiments were based on protocols available from the manufacturer [3]. The amylase levels in saliva vary between individuals, and for one person over time. Therefore saliva from two persons was collected on the day of each experiment, and analysed in parallel. To avoid cross contamination, the sampled persons did not take part in the laboratory work. One saliva dilution series (1:1, 1:5, 1:10, 1:25, 1:50, 1:100, 1:200, 1:500) was prepared for each sampled person, using physiological saline-solution as agent. A triplicate of 50 ml of each dilution was dispensed on cotton fabric (T-shirt), together with one negative control (50 ml of physiological salinesolution). For each dilution series, two identical pieces of cotton fabric were prepared for parallel development in room temperature and 37 8C (heating cabinet). The stains were left to dry in room temperature for approximately 2 h before analysis. The pieces of cotton fabric were put on a clean operation cloth and moistened using de-ionised water. A piece of Phadebas1 Forensic Press test paper was put on top of the fabric and moistened to saturate the paper, thereby starting the reaction. A glass plate was put on top of the paper and a weight used to
J. Hedman et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 430–432
press the paper tightly against the fabric. The progress of the reactions was monitored every 5 min. After 40 min the reaction was interrupted, and the paper left to dry. Six different carrier materials were studied: denim, cotton fabric (T-shirt), leather, suede, untreated wood and painted wood. Two saliva dilution series were prepared (1:1, 1:5, 1:25, 1:100). Triplicates of 50 ml of each dilution were dispensed on each set of materials, together with one negative control. The exhibits were left to dry over night, and studied using four different lightsources: Crime Scope1 CS-16-10, 430 nm/ yellow goggles (Spex); Quaser 2000/30, 340-413 nm/transparent goggles (Foster & Freeman); UV spotlight, 365 nm/ transparent goggles (Labino) and Polilight1 PL6, 425 nm/ yellow goggles (Rofin). Phadebas1 development was performed approximately 24 h after application of the stains, following the protocol described above (room temperature). After drying the exhibits and the Phadebas1 paper, sampling for DNA analysis was performed. Two stains, of two chosen saliva concentrations (1:1 and the lowest concentration showing a positive Phadebas1 result), from each exhibit and the corresponding Phadebas1 paper were sampled. The stains on untreated wood, painted wood, leather and suede were swabbed using cotton swabs, cut and placed in microfuge tubes. The stains on cotton fabric, denim and the Phadebas1 papers were cut directly (1.5 cm 1.5 cm) into microfuge tubes. Chelex 1001 extraction was used for all samples [4]. For leather and suede a step of Centricon1-100 tube purification was performed [5]. The DNA analysis was performed using Applied Biosystems 7300 Real Time PCR System with QuantifilerTM Human DNA Quantification Kit, GeneAmp1 PCR System 9700 with AmpFISTR1 SGM Plus1 PCR Amplification Kit, ABI PRISM1 3130xl Genetic Analyzer and GeneMapperTM ID v3.1. 3. Results and discussion With pure saliva, strong colour changes could be seen for all stains within 5 min. After 40 min, stains were visible down to dilution 1:25 for both room temperature series and one 37 8C series. Weak results were observed for dilution 1:100 at room temperature and for 1:200 at 37 8C (one series). The second 37 8C series only gave results to 1:10. After drying of the Phadebas1 paper, strong results could be seen down to dilution 1:50 at room temperature. For all positive results, blue colour from the paper was displaced onto the cotton fabric, generating a visible detection on the examined exhibits. No results were seen for the negative controls. The inconsistency for incubation in 37 8C is probably caused by elevated evaporation, and the problem to moisten the material and paper evenly and enough. Since room temperature showed more even results, and is handier to apply both at crime scenes and in the laboratory, this was chosen for the remaining experiments. However, the indications of lower detection levels at higher temperatures should be evaluated further. On denim, no stains could be detected using the lightsources, not even for pure saliva. Using Phadebas1, both the 1:1 and 1:5 dilutions were clearly visible after 40 min of incubation. On
431
cotton fabric, Phadebas1 gave results down to 1:100, whereas the lightsources failed for pure saliva. However, all lightsources except Crime Scope1 visualised stains for the saline negative controls, indicating that something else than saliva was fluorescing. For untreated wood, the UV spotlight gave fluorescence for all stains including the negative control, whereas the other lightsources failed to visualise any stains. Phadebas1 showed clear stains down to dilution 1:100. On leather, Phadebas1 and the UV spotlight gave results down to 1:25, the Quaser only managed concentrated saliva. The other lightsources failed to visualise any stains. For painted wood, the lightsources visualised stains down to 1:25, whereas Phadebas1 clearly showed all stains down to 1:100. On suede, Phadebas1 showed no stains, whereas Crime Scope1 and UV spotlight gave results down to 1:25 and 1:100, respectively. However, Crime Scope1 visualised stains for the saline negative control as well. The Phadebas1 test generated better results than any lightsource for all materials, except for suede. The comparative advantage of the saliva specific amylase method versus the lightsources is obvious. However, the longer detection time and invasive character of the method might benefit use of lightsources. It is clear that highly absorbing surfaces, such as suede and denim, are troublesome for both amylase based and lightsource detection, as the saliva appears trapped inside the texture of these exhibits. Still, Phadebas1 seemed to work better on these material types. It is clear that it is very important to moisturise the Phadebas1 paper and the exhibit well, especially for highly absorbing materials. Some problems during this project emanated from not using enough water and this has to be considered in further studies. The DNA analyses of pure saliva stains on different materials generated complete profiles in all cases tested. For leather and painted wood, the DNA analyses of the corresponding piece of Phadebas1 paper also yielded complete profiles, and the amount of DNA was approximately the same as from the exhibits. For the other exhibits, the paper samples failed to produce any analysable amounts of DNA. The plausible reason for this is that less absorbent or inert exhibits are more prone to transfer cells to the Phadebas1 paper. Of the samples from the lowest detected stains, only the 1:5 dilutions (denim and leather) gave enough DNA to enable analysis. This suggests that the Phadebas1 test is more sensitive for saliva stain detection than the standard methods for forensic DNA analysis. The 1:100 dilutions that were successfully detected on some materials correspond to 0.5 ml pure saliva in the 50 ml stain, which is a very minute amount when it comes to DNA analysis. The detection levels for Phadebas1 are also highly dependent on the nature of the background material. No PCR inhibition could be seen in the analysis, suggesting that the Phadebas1 chemistry does not interfere with the subsequent PCR. In conclusion, the Phadebas1 Forensic Press test gives a good indication to whether or not a crime scene stain contains saliva suitable for DNA analysis. However, positive stains might contain too little DNA and negative stains might produce complete DNA profiles.
432
J. Hedman et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 430–432
Conflict of interest None. References [1] G.M. Willot, An improved test for the detection of salivary amylase in stains, J. Forensic Sci. Soc. 14 (1974) 341–344.
[2] G.M. Willott, M. Griffiths, A new method for locating saliva stains—Spotty paper for spotting spit, Forensic Sci. Int. 15 (1980) 79–83. [3] Magle Life Sciences. http://www.phadebas.com. [4] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 1001 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (4) (1991) 506–513. [5] Millipore. http://www.millipore.com.