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Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three weeks after insemination. During early pregnancy, the expression of interferon-stimulating genes (ISGs) in peripheral blood leukocytes (PBL) is significantly increased. However, due to different strains, detection sample types, detection methods, threshold value, etc. the specific effectiveness of early pregnancy diagnosis using ISGs is worth further study. The purpose of this study was to test interferon-stimulated protein 15 ( ISG15 ), 2'–5'-oligoadenylate synthetase 1 ( OAS1 ) and radical S-adenosyl methionine domain containing 2 ( RSAD2 ) for early pregnancy diagnosis in dairy cows. The expression of  ISG15 ,  OAS1 , and  RSAD2  in PBL of pregnant and non-pregnant heifers on days 0, 14, 18, 21 and 28 after artificial insemination (AI) was detected by fluorescence quantitative polymerase chain reaction (PCR). The sensitivity and specificity of the pregnancy diagnosis was analyzed using expression of these three genes separately or in combination by receiver operating characteristic curve. The combination with the highest accuracy used probe primers and duplex fluorescence quantitative PCR. The single quantitative PCR results showed that expression of  ISG15 ,  OAS1  and  RSAD2  on day 18 after AI was significantly higher in pregnant than in non-pregnant cows. When these three genes were used separately, or in combination, for early pregnancy diagnosis, the sensitivity for the  RSAD2  gene was 100%, and the combination of  ISG15  with  RSAD2  was 94.7%. The duplex quantitative PCR showed that, although the sensitivity of  ISG15  alone was 100%, its specificity was only 88.2% (cut-off value 1.402). The sensitivity of  RSAD2  alone was 89.5%, and the specificity was 88.2%; however, when the two genes were used in combination, the sensitivity, specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR. These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using  ISG15  and  RSAD2  was established. Simultaneous detection of expression of  ISG15  and  RSAD2  by duplex quantitative PCR can effectively improve the diagnostic accuracy for dairy cows.

Duplex Quantitative Polymerase Chain Reaction of ISG15 and RSAD2 Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows