DNA Profiling of Horse Urine Samples to Confirm Donor Identity RIRDC Publication No. 09/076
RIRDC
Innovation for rural Australia
DNA Profiling of Horse Urine Samples to Confirm Donor Identity
by Paula Hawthorne, Jenny Wang-Holmes, Judy Cawdell-Smith and Ann E.O. Trezise
July 2009 RIRDC Publication No 09/076 RIRDC Project No PRJ-000522
© 2009 Rural Industries Research and Development Corporation. All rights reserved.
ISBN 1 74151 876 8 ISSN 1440-6845 DNA Profiling of Horse Urine Samples to Confirm Donor Identity Publication No. 09/076 Project No. PRJ-000522 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.
Researcher Contact Details Assoc. Professor Ann Trezise Level 7, Chemistry Building (68) Cooper Rd University of Queensland St Lucia QLD 4072 Phone: 07 3365 3647 Fax: 07 3365 4899 Email:
[email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web:
02 6271 4100 02 6271 4199
[email protected]. http://www.rirdc.gov.au
Electronically published by RIRDC in July 2009 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313
ii
Foreword There are approximately 20 000 horse races per year in Australia, equating to an average of 60 races per day (Australian Racing Board, 2001). Three to five horses from each race may be randomly sampled for drugs. Formal hearings involving positive drug tests are conducted and frequently the source of the sample that tested positive is challenged. The capacity to provide independent and unambiguous confirmation of the identity of the donor of a drug-positive urine sample would be of enormous benefit to the Australian horse racing industry. It would remove any question of sample substitution, and would provide unequivocal identification of the horse that provided the sample. Confirmation of identity of a donor horse can be achieved by DNA profiling. Human forensics uses DNA profiling by amplification of sets of DNA microsatellite markers to confirm identity. Similarly, the identity of horses can also be confirmed by DNA profiling of sets of microsatellite markers. Horses of many different breeds are currently DNA profiled for breeding and registration purposes. DNA for these purposes is usually extracted from horse blood or hair samples. DNA profiling from urine has been achieved in human forensics and for some horse samples, but the method has been consistently problematic. This is due to a number of factors such as the small amount of DNA in urine samples, differences in male and female samples, presence of inhibitors to amplification, and rapid degradation of the DNA in urine samples. The research in this project aimed to investigate methodologies to consistently obtain reliable DNA profiles from horse urine samples and to investigate how a process for DNA profiling of urine samples could be integrated with horse racing industry drug testing laboratories across Australia. This report, an addition to RIRDC’s diverse range of over 1800 research publications, forms part of our Horse R&D program, which aims to assist in developing the horse industry and enhancing its export potential. This project was funded from RIRDC Core Funds which are provided by the Australian Government, in conjunction with funds from industry. Most of RIRDC’s publications are available for viewing, downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.
Peter O’Brien Managing Director Rural Industries Research and Development Corporation
iii
Acknowledgments We would like to thank the Rural Industries Research and Development Corporation (Horse R&D Program) for funding this research, the Australian Stud Book, which funded the industry component of the research, as well as our other collaborators, the University of Queensland (School of Animal Studies and School of Biomedical Sciences), Racing Science Centre (Office of Racing, Queensland Treasury) and Australian Racing Forensic Laboratory (Racing NSW).
Abbreviations AEGRC
Australian Equine Genetics Research Centre
SOP
Standard Operating Procedure
HCl
Hydrochloric acid
iv
Contents Foreword ...............................................................................................................................................iii Acknowledgments................................................................................................................................. iv Abbreviations........................................................................................................................................ iv Executive Summary.............................................................................................................................. vi Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 2 Methodology........................................................................................................................................... 3 Results..................................................................................................................................................... 4 Implications............................................................................................................................................ 8 Recommendations.................................................................................................................................. 9 References ............................................................................................................................................ 10
v
Executive Summary What the report is about The capacity to provide independent and unambiguous confirmation of the identity of the donor of a biological test sample (whether urine or blood) would be of enormous benefit to the Australian horse racing industry. There are approximately 20 000 horse races per year in Australia, equating to an average of 60 races per day (Australian Racing Board, 2001). Drug testing is routinely carried out in race meets across Australia and has become an increasingly sophisticated science with tests being made for an increasing number of drugs. However, the increased efficiency and frequency of drug testing has also led to a need for independent confirmation of the identity of a drug-positive urine sample. Frequently, the identity of the donor of a drug-positive urine sample is challenged. Independent confirmation would remove any question of sample substitution and would provide unequivocal identification of the horse that provided the drug-positive sample. This is the final report for a RIRDC-funded research project that aimed to develop methods to allow urine samples from horses to be independently identified using DNA profile analysis. Who is the report targeted at? This report is for the RIRDC Equine Science Panel. The report also makes recommendations that are relevant to Australian horse racing authorities, Australian horse racing drug testing laboratories, and the horse racing, harness racing, turf and jockey clubs and authorities across Australia. Background Confirmation of identity and parentage by DNA profiling is routinely carried out in the Australian horse industry and around the world by DNA profiling using sets of DNA microsatellite markers. This confirmation of identity is mostly used for horse breeding and registration purposes and import and export purposes. DNA for profiling is extracted for these purposes mainly from horse hair but sometimes from other samples such as blood or semen. Methodologies to extract DNA from these samples are fairly advanced, with DNA profiling from these samples able to be achieved in short periods of time, usually one to two weeks. DNA profiling from urine has been achieved in human forensics, and in some cases from horses, but extraction of DNA from urine samples in a consistent and reliable manner has been elusive in both human and animal forensics. Extraction of DNA from urine has proved difficult due to the small amount of cellular material in urine and the degraded state of DNA that is extracted from urine. The amount of DNA in urine also shows large variability betweens samples. Urine also contains many substances which inhibit amplification of extracted DNA. The research in this project aimed to investigate methodologies to consistently obtain reliable DNA profiles from horse urine samples and to investigate how a process for DNA profiling of urine samples could be integrated with horse racing industry drug testing processes across Australia. Aims/objectives The specific aims of the project were to: •
test various methods for DNA profiling of horse urine samples
•
establish and optimise a reliable DNA profiling method for determining the donor identity of drug positive horse urine samples
•
validate and refine the procedure for routine operation
vi
•
publicise project outcomes to encourage implementation as a standard operating procedure in the industry.
The ability to identify a donor horse by DNA profiling from a drug-positive urine sample will increase confidence in the handling of drug test samples, and has the potential to reduce hearing costs by providing rapid and unequivocal evidence of the identity of the donor. This project may assist in confirmation of identity of the donor of a drug-positive sample, but more importantly, could provide definitive evidence exonerating a horse and trainer. Also, in collaboration with the Australian Stud Book, the identity of unknown or rare mis-identified samples could be established through querying the Australian Equine Genetics Research Centre (AEGRC) database that holds the registered DNA profiles of all Australian thoroughbred horses. This project will also benefit the racing public through increasing public confidence in the racing industry and demonstrating the integrity of the industry. This research then will specifically benefit owners and trainers of horses, drug testing laboratories, stewards, regulators, horse racing clubs, harness racing clubs, turf clubs, jockey clubs and other officials and organisations involved in equine racing. Methods used Many different methods for DNA extraction were tested on horse urine samples. These included published methods, commercially available kits, unpublished methods communicated from other international equine DNA profiling laboratories, and combinations of all of these. Different volumes of urine were tested with these methods. Effects of storage temperature and time on DNA extraction and amplification success from horse urine samples were also tested. Amplification of extracted DNA for a set of twelve microsatellite markers and analysis of amplification results was carried out according to the (AEGRC) standard operating procedures (SOP’s). This amplification involves a multiplex fluorescent PCR system, capillary electrophoresis and analysis by Genemapper software (Applied Biosystems). The AEGRC’s DNA profiling system for horses has been certified under an ISO9001-accredited quality management system since 2006. Results/key findings The first objective of this project proved the most technically challenging and time consuming. Many DNA extraction methods were initially tested on horse urine samples without success. However, with increasing knowledge of DNA extraction from urine, and newer technology, successful DNA profiling of some horse urine samples has been achieved. Twelve microsatellite markers are routinely used to DNA profile equine samples at the AEGRC. These markers contain nine microsatellites recommended by the International Society of Animal Genetics and three extra markers. Initially, utilising centrifugation, saline washing and a commercially available DNA extraction kit (Gentra Puregene), eight complete markers out of twelve microsatellite markers were able to be amplified from a horse urine sample. These markers matched the DNA profile obtained from a hair sample of the same horse. This study also found some success with DNA extraction from urine using hydrochloric acid (HCl) to remove insoluble calcium carbonate and calcium oxalate. These substances sediment with cellular material and inhibit extraction of DNA. Using this method a DNA profile with nine complete markers out of the twelve microsatellite markers was obtained from a horse urine sample. Again, these markers matched those from a hair sample from the same horse. However, the most successful method used was a recently developed commercially available kit, QIAAmp Micro Kit (Qiagen). Using this kit full DNA profiles with twelve complete markers were amplified.
vii
Storage time and temperature were found to have a significant effect on the DNA profiling of horse urine samples. Successful DNA profiling has only been achieved from samples stored at 4°C for not more than two days and then extracted and profiled, or from urine samples stored at -20°C or -80°C after storage at 4°C for not more than two days. No profiles were obtained from samples that were stored at 4°C for a week or longer. The volume of urine used was also found to have a significant effect on the ability to obtain a DNA profile. Although very small volumes of urine have been reported to give a DNA profile in human studies, the results in this study suggest that at least 1 ml of urine is needed to obtain DNA profiles from horse urine samples. Variability between urine samples seems to be an ongoing issue. In this study we tested seven urine samples. To date, only four of these samples have produced full profiles with twelve complete markers. This variation may have been due to excess handling and repeated freeze-thawing, or it could be due to different shedding rates of epithelial cells between different individuals. In humans, female urine samples are seen to contain more cellular material than male samples, but this has not yet been established for horses. All full profiles obtained in this study were from male horses, but a larger sample set with more female horses is required to establish whether there is a difference in DNA extraction from urine due to the sex of the horse. The methodology of extracting DNA from urine samples has proved the most technically and timeconsuming challenge of this research project. However, important progress has been made; the ability to obtain twelve markers in a DNA profile gives confirmation of identify of origin of a sample with a confidence level of greater than 1 in 100 000 000 000 (1 in 100 billion). In human forensics, with degraded or difficult samples like urine, samples in which the full complement of markers has not been obtained are still used in court cases, as even with a smaller number of markers the power of confirmation of identity with DNA profiling is still very high. Therefore, once this method is optimised further to overcome variability between samples it could be offered as a service to racing drug testing laboratories when the identity of a sample is challenged. The AEGRC has achieved IS09001 certification and therefore has the procedures in place to comply with chain of custody and quality assurance requirements from drug testing laboratories. The AEGRC has DNA profiled blood samples in line with these requirements for a racing drug testing laboratory. The AEGRC participates in International Accuracy Tests and correctly profiled 100% of 500 DNA markers on their last test. Therefore, it is anticipated that once a method is optimised actual integration of the method with procedures of racing drug testing laboratories should be achieved in a very short period of time. Implications for relevant stakeholders: The results of this study provide promise that DNA profiling from urine is close to becoming a procedure that can be offered to confirm the identity of drug-positive samples. However, due to the nature of the urine samples and variability in storage conditions there are issues that will need to be addressed with relevant stakeholders. Australian horse racing drug-testing laboratories will be a major stakeholder for this test. Volumes of urine required and storage conditions of urine will need to be discussed with these laboratories, as these factors will affect their collection procedures. The variable nature of DNA profiling from urine samples must also be discussed with the racing drug testing laboratories. The time consuming technical challenges of determining a reliable method for extracting DNA from urine has meant drug-positive urine samples have not been DNA profiled so far. It is possible that a particular drug may inhibit DNA profiling in some way. The ranges of drugs tested in urine must be discussed with these laboratories and once a final optimised method has been chosen the effect of these drugs on DNA profiling of urine samples should be tested.
viii
If this method for extraction and profiling of DNA from horse urine samples is adopted by racing drug testing laboratories, trainers and owners must be informed about the test being performed and its legal implications for their challenges to the identity of drug-positive samples. While this project may result in confirmation of identity of drug-positive samples, it could also provide evidence exonerating a horse and trainer.
Recommendations Recommendations from the findings of this study are that the AEGRC should: •
continue to optimise the methodology for DNA profiling of urine samples and integrate this methodology into their normal service delivery.
•
collaborate with racing drug testing laboratories to obtain urine samples positive for the complete range of drugs the laboratories test for, and test the DNA extraction and profiling method on these urine samples.
•
collaborate with racing drug testing laboratories to discuss urine sample volume, transportation and storage requirements.
ix
Introduction The capacity to provide independent and unambiguous confirmation of the identity of the donor of a urine sample would be of enormous benefit to the Australian horse racing industry. There are approximately 20 000 horse races per year in Australia, equating to an average of 60 races per day (Australian Racing Board, 2001). Drug testing is routinely carried out in race meets across Australia and has become an increasingly sophisticated science, with an increasing number of drugs being tested for. However, the increased efficiency and frequency of drug testing has also led to a need for independent confirmation of the identity of drug-positive urine samples. Frequently, the identity of the donor of a drug-positive urine sample is challenged. Independent confirmation would remove any question of sample substitution, and would provide unequivocal identification of the horse that provided the drug- positive sample. Confirmation of identity by DNA profiling is routinely carried out in the Australian horse industry and around the world by DNA profiling using sets of DNA microsatellite markers. This confirmation of identity is mostly used for horse breeding and registration purposes and import and export purposes. DNA for profiling is extracted for these purposes mainly from horse hair but sometimes from other samples such as blood or semen. Methodologies to extract DNA from these samples are fairly advanced with profiling from these samples able to be achieved in short periods of time. DNA profiling from urine has been achieved in human forensics (Tsongalis et al., 1996; Marques et al., 2005) and in some cases from horses (Marklund et al., 2006; Tobe et al., 2007), but extraction of DNA from urine samples in a consistent and reliable manner has been elusive in both human and animal forensics. This is due to a number of factors including the nature of the urine sample, storage conditions and the amount of DNA in urine. Extraction of DNA from urine has proven difficult due to the small amount of cellular material in urine and degradation of the DNA that is extracted from urine. The cellular component of urine mostly originates from shedding of epithelial cells lining the urethral and bladder tracts. This cellular component is a very small component of a total urine sample and is subject to rapid degradation due to the acidic nature and presence of salts in urine. As intact cells in urine undergo cell death, nucleases are released which break down DNA. The amount of cellular material in urine also shows large variability between samples. It has been observed that human female urine samples consistently contain more cells and therefore more DNA than human male samples (Nakazono et al., 2005; Johnson et al., 2007). However, this has not been established for horse urine samples. Urine also contains a number of substances such as urea which have been shown to inhibit amplification of DNA extracted from urine (Khan et al., 1991). More consistent success with extraction and amplification has been obtained with mitochondrial DNA from urine samples as opposed to nuclear DNA. The amount of mitochondrial DNA may exceed by 1 000 times the number of nuclear DNA copies in mammalian cells (Bogenhagen & Clayton, 1974). However, effort has been concentrated on nuclear DNA extraction and amplification, as identity cannot be determined from mitochondrial DNA, only identification of a maternal lineage. A reliable method for DNA extraction and profiling from urine samples with outputs as consistent as those of DNA extraction and profiling from other samples such as blood, hair and semen, would provide enormous benefit to the Australian horse racing industry. If identity of donor urine samples could easily be determined in a quality-assured manner, challenges to the identity of samples would likely reduce, as would the associated costs. The ability to determine the identity of a donor urine sample would also provide evidence of the quality assurance processes of racing drug testing laboratories, increasing confidence among the stakeholders in the industry. The research in this project aimed to investigate methodologies to consistently obtain reliable DNA profiles from horse urine samples and to investigate how a process for DNA profiling of urine samples could be integrated with racing drug testing laboratories across Australia.
1
Objectives The specific aims of the project were to: •
test various methods for DNA profiling of horse urine samples
•
establish and optimise a reliable DNA profiling method for determining the donor identity of drug positive horse urine samples
•
validate and refine the procedure for routine operation
•
publicise project outcomes to encourage implementation as a standard operating procedure in the industry.
2
Methodology Seven urine samples, provided by collaborators from the School of Animal Studies at the University of Queensland, were tested. A reference hair sample was provided with each urine sample. For each sample the reference hair was extracted and DNA was profiled using the SOP’s of the AEGRC which utilise 12 DNA microsatellite markers for identity analysis. These markers contain nine microsatellites recommended by the International Society of Animal Genetics and three extra markers. This combination of markers is also used internationally by the Veterinary Genetics Laboratory at the University of California, Davis, USA. The chance of two unrelated horses having identical DNA profiles utilising this set of 12 markers is greater than 1 in 100 000 000 000 (the ‘match probability’ statistic; Bowling, 2001). After analysis the DNA profiles of the reference hair samples were stored in the AEGRC database. Urine samples were transported at a temperature of 4°C. Each urine sample was approximately 100ml volume. The urine samples were halved, and half the sample was stored at -20° C or -80°C, the other half of the sample was stored at 4°C. DNA extraction was attempted on all urine samples using various methods including commercially available kits (e.g. Gentra Puregene Tissue Core Kit A, MoBio DNA Extraction Kit, Qiagen Spin Miniprep Kit, QIAAmp DNA Micro Kit), published DNA extraction protocols, unpublished methods, or combinations of all methods. Urine was mostly prepared for DNA extraction by centrifugation to extract cellular material and the supernatant removed. Various methods involved refinements to urine preparation such as the addition of HCl to remove insoluble calcium chloride or calcium oxalate. These substances sediment with cellular material and inhibit extraction of DNA (Harper C, Guthrie A, pers comm.). DNA extraction using commercially available kits was performed according to the manufacturer’s instructions. Different volumes of urine were tested with different extraction methods. Amplification of extracted DNA from urine samples was performed according to the SOP’s of the AEGRC.
3
Results The first objective of this project proved the most technically challenging and time consuming. Many DNA extraction methods were initially tested on horse urine samples without success. However, with increasing knowledge of DNA extraction from urine, and newer technology, successful DNA profiling of some horse urine samples was achieved. Initially, by utilising centrifugation of the cellular material in the urine, saline washing of the recovered cellular material and a commercially available DNA extraction kit (Gentra Puregene), eight complete markers out of twelve microsatellite markers were able to be amplified from a horse urine sample. These markers matched the DNA profile from the reference hair sample of the same horse (Table 1). Successful DNA profiling with another urine sample, obtaining nine complete markers out of the twelve microsatellite markers, was achieved with a method involving addition of HCl to remove insoluble calcium carbonate and calcium oxalate, which are known to inhibit the extraction of DNA. Again these markers matched with the reference hair sample from the same horse (Table 2). Other markers were obtained from some of the other remaining urine samples with this method, with the number of markers amplified ranging from one to five. However, obtaining the full twelve DNA markers from urine samples is not absolutely necessary. The ability to obtain at least eight markers is important progress, as eight DNA markers still gives statistically sufficient confirmation of the identity of a donor sample, with the chance of two unrelated horses having the same eight DNA markers in common being greater than 1 in 100 000 000. Extra markers are required for statistically significant parentage verification, but in the case of drug-positive samples it is individual identity being confirmed, not parentage.
1
Marker Urine Sample Seven
JO
Reference Hair Sample Seven
JO
2
3
4
KK
JK
KK
5
6
JS
GN
JS
IQ
7
8
9
PP
NO
KM
PP
NO
KM
10
MO
11
12
LL
LR
LL
LR
Table 1. Eight markers amplified from DNA extracted from urine utilising a saline wash are identical to those from the hair reference sample. This table details the allele identities (G, I, J, etc) for eight microsatellite markers successfully amplified from DNA extracted from urine treated by centrifugation, saline wash and then extraction with the Gentra Puregene DNA extraction kit. Each microsatellite marker contains two alleles, as each horse receives one allele from each parent. The alleles amplified from urine are identical with those amplified from the same gene markers from the reference hair sample, which was collected from the same horse (Horse 7). The blank spaces in the table indicate either that no amplification result was achieved for a particular microsatellite marker, or that the result was not of sufficient quality to be assigned.
4
1
2
Urine Sample Five
JJ
KK
Reference Hair Sample Five
JJ
KK
Marker
3
4
5
GO
KK
GO
JS
6
7
8
9
10
11
12
PP
PP
OO
KM
IM
M
MM
PP
PP
OO
KM
IM
MQ
MM
Table 2. Nine markers amplified from DNA extracted from urine utilising an acid treatment are identical to those from the hair reference sample. This table details the allele identities (G, I, J, etc) for nine microsatellite markers successfully amplified from DNA extracted from urine treated by addition of HCl, centrifugation and then extraction with the Gentra Puregene DNA extraction kit. Each gene marker contains two alleles, as each horse receives one allele from each parent. The alleles amplified from urine are identical to those amplified from the reference hair sample, which was collected from the same horse (Horse 5). The blank spaces in the table indicate either that no amplification result was achieved for a particular microsatellite marker or that the result was not of sufficient quality to be assigned. In microsatellite marker 11 one allele was amplified, but not the other, so this was not included as a complete marker amplified.
Although full DNA profiles are not necessary for statistical conformation of identity, full DNA profiles were obtained from DNA extracted from horse urine using the QIAAmp DNA Micro Kit (Qiagen), a recently developed commercially available kit. Tobe et al. (2007) reported amplification of 17 microsatellite markers from a horse urine sample using this kit. This kit provided the most consistent results and success in amplifying microsatellite markers. Full DNA profiles were obtained from four of the seven urine samples trialled in this study. Two of these samples were Samples Five and Seven, which had already been used to obtain nearly full profiles using other DNA extraction methods reported in this study (Tables 1 and 2). The other two full profiles obtained are shown in Tables 3 and 4. All DNA profiles obtained from urine samples were identical to the DNA profile from the reference hair sample from each horse. 1
Marker
2
3
4
5
6
7
8
9
10
11
12
Urine Sample Three
JO
JJ
NR
KQ
LT
NN
OP
NN
RR
LM
QQ
IM
Reference Hair Sample Three
JO
JJ
NR
KQ
LT
NN
OP
NN
RR
LM
QQ
IM
Table 3. Twelve markers amplified from DNA extracted from urine utilising the QIAAmp DNA Micro Kit (Qiagen) are identical to those from the hair reference sample. This table details the allele identities (G, I, J, etc) for twelve microsatellite markers successfully amplified from DNA extracted from urine using the QIAAmp DNA Micro Kit (Qiagen). Each microsatellite marker contains two alleles, as each horse receives one allele from each parent. The alleles amplified from urine are identical to those amplified from the reference hair sample, which was collected from the same horse (Horse 3).
5
1
Marker
2
3
4
5
6
7
8
9
10
11
12
Urine Sample Six
HH
JJ
OQ
KM
IS
MP
OO
LN
IR
KK
GQ
NN
Reference Hair Sample Six
HH
JJ
OQ
KM
IS
MP
OO
LN
IR
KK
GQ
NN
Table 4. Twelve markers amplified from DNA extracted from urine utilising the QIAAmp DNA Micro Kit (Qiagen) are identical to those from the hair reference sample. This table details the allele identities (G, I, J, etc) for twelve microsatellite markers successfully amplified from DNA extracted from urine using the QIAAmp DNA Micro Kit (Qiagen). Each microsatellite marker contains two alleles, as each horse receives one allele from each parent. The alleles amplified from urine are identical to those amplified from the reference hair sample, which was collected from the same horse (Horse 6).
The effect of storage time and temperature on the success of DNA extraction and amplification from urine samples was also investigated. Urine samples stored at 4°C and extracted not more than two days after arrival in the laboratory produced successful profiles. DNA extractions from urine samples which were immediately frozen at -20°C or -80°C also resulted in amplification of microsatellite markers. However, extractions from aliquots stored at 4°C for longer than two days produced no marker results when amplified. This is most likely due to released nucleases still being active at this temperature and thus degrading the DNA. It is unlikely nucleases are still active in samples stored at -20°C or -80°C. The volume of urine used was also found to have a significant effect on the ability to DNA profile horse urine samples. In human studies DNA profiling has been achieved from as little as 100 μl of urine (Yasuda et al., 2003) or from urine stains (Nakazono, 2005). In this study volumes of urine ranging from 1 ml to 20 ml were tested, on the basis that horse urine samples may contain less cellular material than human samples, and that greater volumes may thus be needed for successful DNA profiling. Such an assessment was considered consistent with other findings that 10 ml of horse urine was the minimum required for successful DNA profiling (Tobe et al., 2007). However, in this study, using the QIAAmp DNA Micro Kit, 1 ml of horse urine was found to be a suitable volume for obtaining DNA profiles. No volumes less than 1 ml were tested. Variability between urine samples was observed to be an issue in this study. Seven urine samples were tested in total. Only four of the samples produced full profiles with twelve complete markers. However, some of the observed variability may have been due to the extent of handling of samples during testing with different DNA extraction methods. Repeated freeze-thawing of samples may affect DNA quality; a new and larger sample set would enable this potential cause of variation to be tested. Another cause of variability may be different shedding rates of epithelial cells between different individuals. In humans, female urine samples are observed to contain more cellular material than male samples. However this has not yet been established for horses, and may not apply. In this study all samples from which full profiles were obtained were sourced from male horses, but a larger sample set is required to further investigate this issue. Determining the methodology for extracting DNA from urine samples proved the most technically and time consuming challenge of this research project. Thus, only the first objective has been achieved.
6
However, important progress has been made, with the AEGRC’s full profile of twelve DNA microsatellite markers successfully being amplified from urine. The chance of two unrelated horses having identical DNA profiles using this set of 12 markers is greater than 1 in 100 000 000 000 (the ‘match probability’ statistic; Bowling, 2001). In human forensics, with degraded or difficult samples like urine, samples in which the full complement of markers has not been obtained are still used in court cases, as even with smaller number of markers the power of confirmation of identity with DNA profiling is still very high (Michaelis, 2008). The second objective of testing the DNA extraction method on drug positive samples is still to be achieved. The AEGRC is still collaborating on this project with horse racing drug testing laboratories. Therefore, obtaining drug-positive samples to test an optimised DNA extraction from urine samples should not be a problem. Once this method is optimised and tested on drug-positive samples it would be hoped to offer this method as a service to racing drug testing laboratories when the identity of a sample is challenged. The AEGRC has achieved IS09001 certification and therefore has the procedures in place to comply with chain of custody and quality assurance requirements from racing drug testing laboratories. The AEGRC has already DNA profiled blood samples in line with these requirements for a racing drug testing laboratory. The AEGRC participates in International Accuracy Tests and correctly profiled 100% of 500 DNA markers on their last test. Therefore, it is anticipated that once a method is optimised, actual integration of the method with procedures of racing drug testing laboratories should be achieved in a very short period of time. Thus, the third and fourth objectives should be the least time consuming of the objectives.
7
Implications This study has provided further proof, consistent with published literature, that DNA can be extracted and profiled from urine samples. As seen in this study and in the literature, variation between urine samples of different individuals remains a major issue. However, technology has progressed and DNA extraction of urine samples is becoming more reliable with newer techniques. The results of this study provide promise that DNA profiling from horse urine samples is close to becoming a procedure that can be offered to confirm the identity of drug-positive samples. However, due to the nature of the urine samples, there are issues that will need to be addressed with relevant stakeholders. Australian racing drug testing laboratories will be a major stakeholder for a procedure of this sort. Volumes of urine required and storage conditions of urine will need to be discussed with these laboratories as these factors will affect their collection procedures. The variable nature of DNA profiling from urine samples must also be discussed with the drug testing laboratories. The time consuming technical challenges of determining a reliable method for extracting DNA from urine has also meant drug-positive urine samples have not been DNA profiled so far. It is possible that a particular drug may inhibit DNA profiling in some way. The ranges of drugs tested in urine must be discussed with drug testing laboratories, and once a final optimised urine DNA profiling method has been chosen, the effect of these drugs on DNA profiling of urine samples should be tested. If this method for profiling of DNA urine samples is adopted by drug testing laboratories, trainers and owners must be informed about the test being performed and its legal implications for their challenges to the identity of drug positive samples. While this project may result in confirmation of identity of drug positive samples, it could also provide evidence exonerating a horse and trainer.
8
Recommendations Recommendations from the findings of this study are that the AEGRC should: •
continue to optimise the method for DNA extraction and profiling from horse urine samples and integrate this method into their normal service delivery;
•
collaborate with racing drug testing laboratories to obtain urine samples positive for the complete range of drugs the laboratories test for, and test our DNA profiling method on these samples;
•
collaborate with racing drug testing laboratories to discuss urine sample volume, transportation and storage requirements.
9
References Australian Racing Board (2001) Size and Scope of the Australian Thoroughbred Racing Industry. Bogenhagen D, Clayton DA (1974) The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. J Biol Chem. 249: 7991-7995. Bowling AT (2001) Historical development and application of molecular genetic tests for horse identification and parentage control. Livestock Production Science 12: 111-116. Johnson DJ, Calderaro AC, Roberts, KA (2007) Variation in nuclear DNA concentrations during urination. J Forensic Sci. 52 (1): 110-113. Khan G, Kangro HO, Coates PJ, Heath RB (1991) Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol. 44: 360-365. Marklund S, Sandberg K, Andersson L (1996) Forensic tracing of horse identities using urine samples and DNA markers. Anim. Biotechnol. 7 (2): 145-153. Marques MAS, Damasceno LMP, Pereira JMG, Caldeira CM, Dias BBFP, Vargens D (2005) DNA typing: an accessory evidence in doping control. J Forensic Sci 50(3): 587-592. Michaelis RC, Flanders RG, Wulff PH (2008) A Litigator’s Guide to DNA: From the laboratory to the courtroom. Elsevier, USA. Nakazano T, Kashimura S, Hayashiba Y, Jara K, Miyoshi A (2005) Successful DNA typing of urine stains using a DNA purification kit following dialfiltration. J Forensic Sci. 50(4): 860-864. Tobe SS, Reid SJ, Linacre AT (2007) Successful DNA typing of a drug positive sample from a race horse. Forensic Science International 173: 85-86. Tsongalis GJ, Anamani DE, Wu AHB (1996) Identification of urine specimen donors by the PM+DQA1 amplification and typing kit. J Forensic Sci 41: 1031-1034. Yasuda T, Iida R, Takeshita H, Ueki M, Nakajima T, Kaneko Y, Mogi K, Tsukahara T, Kishi K. (2003) A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit. J Forensic Sci. 48(1): 108-110.
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DNA Profiling of Horse Urine Samples to Confirm Donor Identity RIRDC Publication No. 09/076 By Ann E.O. Trezise
The capacity to provide independent and unambiguous confirmation of the identity of the donor of a biological test sample (whether urine or blood) would be of enormous benefit to the Australian horse racing industry. Drug testing is routinely carried out in race meets across Australia and has become an increasingly sophisticated science with tests being made for an increasing number of drugs. However, the increased efficiency and frequency of drug testing has also led to a need for independent confirmation of the identity of a drug-positive urine sample. This would remove any question of sample substitution and would provide unequivocal identification of the horse that provided the drugpositive sample.
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