BACKGROUND: Heparin is a sulfated glycosaminoglycan. Blood is a common source for DNA detection in all kinds of samples, and anticoagulants such as heparin and ethylenediaminetetraacetic acid (EDTA) are used to prevent coagulation. Because heparin has a strong inhibitory effect on polymerase chain reaction (PCR), it is not used in samples that will be tracked by DNA. There are physical, chemical, and enzymatic methods to eliminate the inhibitory effect of heparin on PCR test.OBJECTIVES: First, to compare the intensity of the inhibitory effect of two anticoagulants, heparin, and EDTA, on the Real-Time PCR (qPCR), and then to investigate the impact of the heparinase enzyme present in the medium culture extract of Pseudomonas aeruginosa, on removing the inhibitory effect of heparin during the real-time PCR.METHODS: In the present study, two blood samples containing heparin and EDTA were subjected to a real-time PCR test to check the intensity of the inhibitory effect. Then, the medium culture extract of Pseudomonas aeruginosa was added to the heparinized blood sample infected with Escherichia coli bacteria in two groups with different conditions. In the first group, the DNA in the heparinized blood sample was extracted by the phenol-chloroform isoamyl alcohol method. Then, these samples were incubated with the extract of Pseudomonas aeruginosa bacteria culture medium at different hours, but in the second group, the samples were incubated at different hours before DNA extraction. Also, the DNA concentration in both groups was measured by a Nanodrop device, and finally, all samples were subjected to a real-time PCR test.RESULTS: The results of the research samples showed that although the heparinized blood sample contains more DNA concentration than the EDTA blood sample, it completely prevents genome replication. Also, incubating heparinized blood with Pseudomonas aeruginosa culture medium extract before DNA extraction for more than 24 hours removes the inhibitory effect of heparin during the real-time PCR, even at a lower cycle threshold than the EDTA-containing sample.CONCLUSIONS: The Pseudomonas aeruginosa culture medium extract may enable researchers to use heparinized blood samples for genome amplification and diagnosis without using expensive and limited commercial heparinase enzyme.

In the present study, the expression pattern of OAS-1 and MX-2 gene in peripheral blood mononuclear cells (PBMC) was associated with the early pregnancy in cattle. A total of 60 animals were selected and divided into 2 groups, treatment (50) and control (10) group and synchronized using double PGF2α protocol by 11 days apart followed by insemination at 72 and 96 hrs after second dose of PGF2α. The cows were subjected to blood collection on day 0, 14, 18, 20 and 25 post insemination and Peripheral Blood Mononuclear Cells (PBMC) were harvested using Histopaque® solution, followed by RNA isolation and cDNA synthesis. A significantly (P≤0.01) higher expression of OAS-1 and MX-2 gene was observed on days 18 and 20 post oestrum by quantitative real-time PCR and concentration of MX-2 protein in serum were significantly higher (P≤0.05) on day 18, 20 and 25 in pregnant cows when compared with that of non-pregnant cows. Hence, the present study is concluded that the expression of OAS-1 and MX-2 genes and their encoded proteins may be used to develop a marker for early pregnancy diagnosis in cattle.