Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.
Abstract: Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.
© 2020 Wiley-VCH GmbH.
Publication Date: 2020-12-14 PubMed ID: 32978850DOI: 10.1111/rda.13831Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research article reports on the successful use of TaqMan duplex real-time PCR, a form of gene detection, to accurately determine the sex of unborn horses. The technique is based on genetic markers in the blood of pregnant mares and could be a beneficial tool for horse breeding.
Research Purpose
The purpose of the study was to develop a non-invasive, quick, and precise method to determine the sex of unborn foetuses in horses. The researchers aimed to use the TaqMan duplex real-time PCR technique, which utilizes genetic markers to identify male or female foetuses.
Methodology
- The study involved 50 pregnant Arabian mares, each pregnant with a single foetus.
- Peripheral blood samples were taken from these mares, and cell-free foetal DNA was extracted from the maternal plasma.
- Duplex real-time PCR assays were then performed on this DNA using TaqMan probes and primers that targeted specific regions of the SRY and TSPY genes, which are indicative of male sex.
- The GAPDH gene was also amplified as a control measure to ensure the accuracy of the DNA extraction procedure.
Results
- Out of the 50 mares, 28 were found to be pregnant with female foetuses and 22 with male foetuses.
- The results were conclusive for 46 of the samples, and from these, 43 were accurately predicted.
- The test demonstrated a sensitivity of 90.48%, a specificity of 96%, and an accuracy of 93.48%.
Conclusions
- Overall, the study concluded that TaqMan duplex real-time PCR is a reliable method for non-invasively determining foetal sex in horses.
- Beyond its effectiveness, the technique exhibited rapid results and lowered the instance of false-positive and false-negative findings.
- As the procedure only involves a simple blood sample collection, it could be integrated seamlessly into routine farming operations and greatly assist breeders.
Cite This Article
APA
Kadivar A, Rashidzadeh H, Davoodian N, Nazari H, Dehghani Tafti R, Heidari Khoei H, Seidi Samani H, Modaresi J, Ahmadi E.
(2020).
Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.
Reprod Domest Anim, 56(2), 287-291.
https://doi.org/10.1111/rda.13831 Publication
Researcher Affiliations
- Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
- Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
- Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
- Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
- Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
- Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
- Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
- Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
- Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
- Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
MeSH Terms
- Animals
- Cell-Free Nucleic Acids / blood
- Female
- Fetus
- Genes, sry
- Genetic Testing / veterinary
- Horses
- Male
- Pregnancy
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
- Sensitivity and Specificity
- Sex Determination Analysis / methods
- Sex Determination Analysis / veterinary
Grant Funding
- 97GRN1M1576 / Shahrekord University
References
This article includes 26 references
- Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS. Implementing prenatal diagnosis based on cell-free fetal DNA: Accurate identification of factors affecting fetal DNA yield. PLoS One 6 e25202.
- Chan KCA, Zhang J, Hui ABY, Wong N, Lau TK, Leung TN, Lo KW, Huang DWS, Lo YMD. Size distributions of maternal and fetal DNA in maternal plasma. Clinical Chemistry 50 88-92.
- Chou Q, Russell M, Birch DE, Raymond J, Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Research 20 1717-1723.
- Clausen FB, Krog GR, Rieneck K, Dziegiel MH. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit. Prenatal Diagnosis 27 6-10.
- Curran S, Ginther O. Ultrasonic diagnosis of equine fetal sex by location of the genital tubercle. Journal of Equine Veterinary Science 9 77-83.
- de Leon PMM, Campos VF, Dellagostin OA, Deschamps JC, Seixas FK, Collares T. Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA). Theriogenology 77 694-698.
- Hill M, Finning K, Martin P, Hogg J, Meaney C, Norbury G, Daniels G, Chitty LS. Non-invasive prenatal determination of fetal sex: Translating research into clinical practice. Clinical Genetics 80 68-75.
- Holder R. Equine fetal sex determination between 55 and 150 days. In Equine reproduction (2nd ed., Vol. 2, pp. 2080-2093) Wiley-Blackwell.
- Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5'--3'exonuclease activity of thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America 88 7276-7280.
- Jorgez CJ, Dang DD, Simpson JL, Lewis DE, Bischoff FZ. Quantity versus quality: Optimal methods for cell-free DNA isolation from plasma of pregnant women. Genetics in Medicine 8 615.
- Kadivar A, Hassanpour H, Dehcheshmeh JA. A novel approach to prenatal fetal sex diagnosis by detecting an insertion in the Y-chromosome of ovine Amelogenin gene. Small Ruminant Research 123 218-223.
- Kadivar A, Hassanpour H, Mirshokraei P, Azari M, Gholamhosseini K, Karami A. Detection and quantification of cell-free fetal DNA in ovine maternal plasma; use it to predict fetal sex. Theriogenology 79 995-1000.
- Kadivar A, Tafti RD, Khoei HH, Nasirabadi MH, Esfandabadi NS, Cheraghi N, Davoodian N. Developing a nested real-time PCR assay for determining equine fetal sex prenatally. Journal of Equine Veterinary Science 40 74-79.
- Li Y, Zimmermann B, Rusterholz C, Kang A, Holzgreve W, Hahn S. Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Clinical Chemistry 50 1002-1011.
- Liang D, Lv W, Wang H, Xu L, Liu J, Li H, Hu L, Peng Y, Wu L. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenatal Diagnosis 33 409-415.
- Lo YD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. The Lancet 350 485-487.
- Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK, Wainscoat JS, Johnson PJ, Chang AMZ, Hjelm NM. Quantitative analysis of fetal DNA in maternal plasma and serum: Implications for noninvasive prenatal diagnosis. The American Journal of Human Genetics 62 768-775.
- Mazloom AR, Džakula Ž, Oeth P, Wang H, Jensen T, Tynan J, van den Boom D. Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma. Prenatal Diagnosis 33 591-597.
- Minárik G, Nagyová E, Repiská G, Szemes T, Vlková-Izrael B, Celec P. Detection of contamination in noninvasive prenatal fetal gender test. arXiv 1510.09009 [q-bio.QM].
- Mortarino M, Garagiola I, Lotta L, Siboni S, Semprini A, Peyvandi F. Non invasive tool for foetal sex determination in early gestational age. Haemophilia 17 952-956.
- Neal H. Determining the sex of equine fetuses: An update. Practice 41 34-39.
- Pan X, Zhang C, Li X, Chen S, Ge H, Zhang Y, Chen F, Jiang H, Jiang F, Zhang H, Wang W, Zhang X. Non-invasive fetal sex determination by maternal plasma sequencing and application in X-linked disorder counseling. The Journal of Maternal-Fetal & Neonatal Medicine 27 1829-1833.
- Perlado-Marina S, Bustamante-Aragones A, Horcajada L, Trujillo-Tiebas M, Lorda-Sanchez I, Ruiz Ramos M, Plaza J, Rodriguez de Alba M. Overview of five-years of experience performing non-invasive fetal sex assessment in maternal blood. Diagnostics 3 283-290.
- Repiská G, Sedláčková T, Szemes T, Celec P, Minárik G. Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma. Clinical Chemistry and Laboratory Medicine 51 1185-1189.
- Resende H, Carmo M, Ramires Neto C, Alvarenga MA. Determination of equine fetal sex by Doppler ultrasonography of the gonads. Equine Veterinary Journal 46 756-758.
- Vincze B, Gáspárdy A, Biácsi A, Papp ÉÁ, Garamvölgyi L, Sós E, Cseh S, Kovács G, Pádár Z, Zenke P. Sex determination using circulating cell-free fetal DNA in small volume of maternal plasma in elephants. Scientific Reports 9 1-7.
Citations
This article has been cited 0 times.Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
