title: A low-cost efficient multiplex PCR for prenatal sex determination in bovine fetus using free fetal DNA in maternal plasma.
abstract: BACKGROUND
  In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction (PCR) analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses.
  
  
  METHODS
  In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 µl maternal plasma. Two primer pairs for bovine amelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome.
  
  
  RESULTS
  The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results.
  
  
  CONCLUSIONS
  The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses.
authors.affiliation: Department of Animal and Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), North Region of Iran, Rasht, Iran ; Department of Medical Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: Arash
authors.initials: A
authors.lastname: Davoudi
authors.affiliation: Department of Animal and Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), North Region of Iran, Rasht, Iran.
authors.forename: Ramin
authors.initials: R
authors.lastname: Seighalani
authors.affiliation: Department of Medical Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
authors.forename: Seyed Ahmad
authors.initials: SA
authors.lastname: Aleyasin
authors.affiliation: Department of Animal and Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), North Region of Iran, Rasht, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Tarang
authors.affiliation: Department of Animal and Poultry Science, College of Aboureyhan, University of Tehran, Tehran, Iran.
authors.forename: Abdolreza Salehi
authors.initials: AS
authors.lastname: Salehi
authors.affiliation: Department of Animal and Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), North Region of Iran, Rasht, Iran.
authors.forename: Farideh
authors.initials: F
authors.lastname: Tahmoressi
keywords: Free Fetal DNA; Maternal Plasma; Multiplex PCR; Sex Determination