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Forensic application of a multiplex PCR system for the typing of pig STRs
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Forensic Science International: Genetics Supplement Series 1 (2008) 614–615 www.elsevier.com/locate/FSIGSS
Research article
Forensic application of a multiplex PCR system for the typing of pig STRs C. Robino a,*, S. Menegon a, S. Caratti a, B. Sona b, S. Gino a, C. Torre a a
Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Corso Galileo Galilei 22, 10126 Turin, Italy b Servizio Veterinario Area Sanita` Animale, ASL 17, Savigliano, Italy Received 17 August 2007; accepted 9 October 2007
Abstract The Animal Type Pig PCR Amplification kit (Biotype AG, Dresden, Germany), a multiplex PCR system that allows the simultaneous amplification of 11 tetrameric short tandem repeats (STRs) and an Amelogenin-like locus for individual identification and sexing of pig (sus scrofa), was employed in the investigation of an alleged case of veterinary malpractice. A veterinarian responsible for collecting random blood samples as part of the program for the serological surveillance of swine vesicular disease (SVD) was accused of having drawn multiple samples from a single sow. STR typing of serum left overs from SVD testing strongly supported the conclusion that all the questioned samples included DNA from the same animal. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Identification; Pig; Short tandem repeat
1. Introduction
2. Materials and methods
In recent years, animal DNA identity and paternity testing by means of PCR-based molecular assays has emerged as an important tool in food safety management and veterinary public health. Here we report the application of a multiplex PCR system for the typing of pig STRs, similar in design to commonly available commercial kits for human STR analysis, in the investigation of an alleged case of veterinary malpractice. A veterinarian responsible for collecting random blood samples from Landrace Large White crossbred sows from a pig farm located in Piedmont (north-west Italy), as part of the program for the serological surveillance of SVD, was accused of having drawn multiple samples from a single animal. Five of the twelve collected samples reacted positively in serological tests for SVD but re-sampling of the apparently infected pigs and their cohort gave completely negative results. Since such a simultaneous seroconversion was extremely unlikely, improper sample collection was suspected. A judicial inquiry was opened and, one year later, DNA identification of samples that had tested positive for SVD virus was ordered.
The five analyzed samples consisted of frozen serum left over from SVD testing, ranging in volume from 200 to 750 ml. Samples were centrifuged for 5 min at 14,000 rpm and the cellular pellet was extracted using the NucleoMag 96 Blood kit (Macherey-Nagel, Du¨ren, Germany) and the automatic magnetic separator KingFisher mL (Thermo Labsystems, Vantaa, Finland). Five microliters aliquots of each DNA extract were directly amplified using the Animal Type Pig PCR amplification kit (Biotype AG, Dresden, Germany), following the manufacturer’s recommendations. Fluorescent PCR products underwent electrophoresis on an ABI Prism Genetic 310 Analyzer (Applied Biosystems, Foster City, CA, USA) and were analyzed by means of software GeneScan 3.7 and Genotyper 3.7 (Applied Biosystems). For statistical evaluation, allele distribution of STRs included in the Animal Type Pig PCR system was determined in a local population sample (n = 55) of Landrace Large White crossbred pigs from Piedmont farms. Random match probability (RMP), under the distinct hypotheses of unrelatedness and relatedness, was calculated based on the formulas elaborated by Weir [1]. Likelihood ratio (LR) for mixtures interpretation was determined using the unrestricted combinatorial approach, according to the recommendations of the International Society of Forensic Genetics [2].
* Corresponding author. Tel.: +39 011 6705625; fax: +39 011 6635576. E-mail address: [email protected] (C. Robino). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.202
C. Robino et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 614–615
615
Table 1 Allele distribution of STR loci in a population sample (n = 55) of Landrace Large White pigs from Piedmont SBH2 5 6 9 10 11 11.1 12 12.1 13 14 14.1 15 15.1 16 16.1 17 17.1 18 19 19.1 20 21 22 23 23.3 24 25 26 28 30 34
SBH18
S0655
SBH1
SBH13
387 A12F
SBH19
SBH22
.091 .336 .036 .209
.036 .027 .300
.273 .009 .045
.082 .045
.100
.300
.064
.418
.173 .027
.255 .482
.027 .064
.109 .036 .418
.018
.145
.109 .145 .073 .136 .055 .164
.127
.027 .327 .291 .273
.027 .100
.018
.036 .036
.027 .009
.009
.091 .009 .245
.118 .055
.009
.136 .064
.045 .482 .055 .073
.009 .009 .336 .009 .027 .100
3. Results and discussion Two of the five questioned samples shared an identical genetic profile. Based on the population data collected in this study (Table 1), the RMP for these two samples resulted to be 1.6 10 13 (under the hypothesis that the two pigs sharing the same genetic profile were unrelated). Taking into account the hypothesis that these samples could come from related individuals, the RMP resulted to be: 9.2 10 6 (full siblings), 5.9 10 10 (half siblings) and 1.7 10 11 (first cousins). The other three samples generated mixed profiles that constantly included the alleles of the above described genetic profile. LRs in favour of the hypothesis that the observed mixtures contained DNA from the individual carrying this profile ranged from 9.9 1011 to 2.8 1013. In conclusion, the obtained results strongly supported the hypothesis that all the different collection tubes contained, exclusively or in part, blood from the same singleton reactor. Many DNA-based systems for animal and parental identification have been recently described for pigs, ranging from single nucleotide polymorphisms (SNPs) to dimeric microsatellites [3]. Population data collected in this study indicate that the multiplex PCR system here described displays a cumulative power of discrimination (>0.999999999) and a probability of exclusion
.009
SBH20 19 20 22 23 29 34 35 36 37 38 39 40 41 42 43 44 45 46 48 49 49.3 50.1 56 56.1 57 58 59 60 60.3 62 66 66.1
.027 .318 .018 .164 .009 .009 .164 .027 .227 .018 .018
SBH10
SBH4
.109 .118 .009 .009 .009 .073 .018 .164 .027 .082 .091 .291 .009 .045 .045 .036 .027 .364 .018 .218 .036 .045 .073 .082
(0.99993) comparable to the above mentioned genetic marker panels. However it must be noted that the analysis of tetrameric STRs offers several advantages in forensic animal genetics, compared to two dinucleotidic microsatellites, because of easier typing (due to the reduced presence of stutter artefacts) and lower mutation rates. Moreover, in selected situations when the identification and interpretation of DNA mixtures is required, as the one reported in this case, microsatellite systems are better indicated than biallelic markers like SNPs. Conflict of interest None. References [1] B.S. Weir, Forensics, in: D.J. Balding, M. Bishop, C. Cannings (Eds.), Handbook of Statistical Genetics, second ed., John Wiley & Sons, Hoboken, NJ, 2003. [2] P. Gill, C.H. Brenner, J.S. Buckleton, et al., DNA commission of the International Society of Forensic Genetics: recommendations on the interpretation of mixtures, Forensic Sci. Int. 160 (2006) 90–101. [3] G.A. Rohrer, B. Freking, D. Nonneman, Single nucleotide polymorphisms for pig identification and parentage exclusion, Anim. Genet. 38 (2007) 253–288.
Forensic Science International: Genetics Supplement Series 1 (2008) 614–615 www.elsevier.com/locate/FSIGSS
Research article
Forensic application of a multiplex PCR system for the typing of pig STRs C. Robino a,*, S. Menegon a, S. Caratti a, B. Sona b, S. Gino a, C. Torre a a
Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Corso Galileo Galilei 22, 10126 Turin, Italy b Servizio Veterinario Area Sanita` Animale, ASL 17, Savigliano, Italy Received 17 August 2007; accepted 9 October 2007
Abstract The Animal Type Pig PCR Amplification kit (Biotype AG, Dresden, Germany), a multiplex PCR system that allows the simultaneous amplification of 11 tetrameric short tandem repeats (STRs) and an Amelogenin-like locus for individual identification and sexing of pig (sus scrofa), was employed in the investigation of an alleged case of veterinary malpractice. A veterinarian responsible for collecting random blood samples as part of the program for the serological surveillance of swine vesicular disease (SVD) was accused of having drawn multiple samples from a single sow. STR typing of serum left overs from SVD testing strongly supported the conclusion that all the questioned samples included DNA from the same animal. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Identification; Pig; Short tandem repeat
1. Introduction
2. Materials and methods
In recent years, animal DNA identity and paternity testing by means of PCR-based molecular assays has emerged as an important tool in food safety management and veterinary public health. Here we report the application of a multiplex PCR system for the typing of pig STRs, similar in design to commonly available commercial kits for human STR analysis, in the investigation of an alleged case of veterinary malpractice. A veterinarian responsible for collecting random blood samples from Landrace Large White crossbred sows from a pig farm located in Piedmont (north-west Italy), as part of the program for the serological surveillance of SVD, was accused of having drawn multiple samples from a single animal. Five of the twelve collected samples reacted positively in serological tests for SVD but re-sampling of the apparently infected pigs and their cohort gave completely negative results. Since such a simultaneous seroconversion was extremely unlikely, improper sample collection was suspected. A judicial inquiry was opened and, one year later, DNA identification of samples that had tested positive for SVD virus was ordered.
The five analyzed samples consisted of frozen serum left over from SVD testing, ranging in volume from 200 to 750 ml. Samples were centrifuged for 5 min at 14,000 rpm and the cellular pellet was extracted using the NucleoMag 96 Blood kit (Macherey-Nagel, Du¨ren, Germany) and the automatic magnetic separator KingFisher mL (Thermo Labsystems, Vantaa, Finland). Five microliters aliquots of each DNA extract were directly amplified using the Animal Type Pig PCR amplification kit (Biotype AG, Dresden, Germany), following the manufacturer’s recommendations. Fluorescent PCR products underwent electrophoresis on an ABI Prism Genetic 310 Analyzer (Applied Biosystems, Foster City, CA, USA) and were analyzed by means of software GeneScan 3.7 and Genotyper 3.7 (Applied Biosystems). For statistical evaluation, allele distribution of STRs included in the Animal Type Pig PCR system was determined in a local population sample (n = 55) of Landrace Large White crossbred pigs from Piedmont farms. Random match probability (RMP), under the distinct hypotheses of unrelatedness and relatedness, was calculated based on the formulas elaborated by Weir [1]. Likelihood ratio (LR) for mixtures interpretation was determined using the unrestricted combinatorial approach, according to the recommendations of the International Society of Forensic Genetics [2].
* Corresponding author. Tel.: +39 011 6705625; fax: +39 011 6635576. E-mail address: [email protected] (C. Robino). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.202
C. Robino et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 614–615
615
Table 1 Allele distribution of STR loci in a population sample (n = 55) of Landrace Large White pigs from Piedmont SBH2 5 6 9 10 11 11.1 12 12.1 13 14 14.1 15 15.1 16 16.1 17 17.1 18 19 19.1 20 21 22 23 23.3 24 25 26 28 30 34
SBH18
S0655
SBH1
SBH13
387 A12F
SBH19
SBH22
.091 .336 .036 .209
.036 .027 .300
.273 .009 .045
.082 .045
.100
.300
.064
.418
.173 .027
.255 .482
.027 .064
.109 .036 .418
.018
.145
.109 .145 .073 .136 .055 .164
.127
.027 .327 .291 .273
.027 .100
.018
.036 .036
.027 .009
.009
.091 .009 .245
.118 .055
.009
.136 .064
.045 .482 .055 .073
.009 .009 .336 .009 .027 .100
3. Results and discussion Two of the five questioned samples shared an identical genetic profile. Based on the population data collected in this study (Table 1), the RMP for these two samples resulted to be 1.6 10 13 (under the hypothesis that the two pigs sharing the same genetic profile were unrelated). Taking into account the hypothesis that these samples could come from related individuals, the RMP resulted to be: 9.2 10 6 (full siblings), 5.9 10 10 (half siblings) and 1.7 10 11 (first cousins). The other three samples generated mixed profiles that constantly included the alleles of the above described genetic profile. LRs in favour of the hypothesis that the observed mixtures contained DNA from the individual carrying this profile ranged from 9.9 1011 to 2.8 1013. In conclusion, the obtained results strongly supported the hypothesis that all the different collection tubes contained, exclusively or in part, blood from the same singleton reactor. Many DNA-based systems for animal and parental identification have been recently described for pigs, ranging from single nucleotide polymorphisms (SNPs) to dimeric microsatellites [3]. Population data collected in this study indicate that the multiplex PCR system here described displays a cumulative power of discrimination (>0.999999999) and a probability of exclusion
.009
SBH20 19 20 22 23 29 34 35 36 37 38 39 40 41 42 43 44 45 46 48 49 49.3 50.1 56 56.1 57 58 59 60 60.3 62 66 66.1
.027 .318 .018 .164 .009 .009 .164 .027 .227 .018 .018
SBH10
SBH4
.109 .118 .009 .009 .009 .073 .018 .164 .027 .082 .091 .291 .009 .045 .045 .036 .027 .364 .018 .218 .036 .045 .073 .082
(0.99993) comparable to the above mentioned genetic marker panels. However it must be noted that the analysis of tetrameric STRs offers several advantages in forensic animal genetics, compared to two dinucleotidic microsatellites, because of easier typing (due to the reduced presence of stutter artefacts) and lower mutation rates. Moreover, in selected situations when the identification and interpretation of DNA mixtures is required, as the one reported in this case, microsatellite systems are better indicated than biallelic markers like SNPs. Conflict of interest None. References [1] B.S. Weir, Forensics, in: D.J. Balding, M. Bishop, C. Cannings (Eds.), Handbook of Statistical Genetics, second ed., John Wiley & Sons, Hoboken, NJ, 2003. [2] P. Gill, C.H. Brenner, J.S. Buckleton, et al., DNA commission of the International Society of Forensic Genetics: recommendations on the interpretation of mixtures, Forensic Sci. Int. 160 (2006) 90–101. [3] G.A. Rohrer, B. Freking, D. Nonneman, Single nucleotide polymorphisms for pig identification and parentage exclusion, Anim. Genet. 38 (2007) 253–288.