Evaluation of physical, chemical, and physicochemical treatments for overcoming the inhibitory effect of heparin on DNA amplification in realtime-PCR

BACKGROUND AND OBJECTIVESHeparin is a highly sulfated glycosaminoglycan used as an injectable anticoagulant. Heparinacts indirectly at multiple sites in both internal and external blood coagulation pathways. Bloodis a common source for tracking DNA samples for various microbial and non-microbial teststo prevent coagulation from EDTA-containing tubes. mg۱ is used per milliliter.Heparin is also a common anticoagulant, but because it has a strong inhibitory effect on PCR,it is not used for samples to be DNA tracked. In all dilutions, heparin inhibits virus replicationin the real-time PCR testMATERIALS AND METHODSIn this study, we intend to investigate different ways to remove the inhibitory effect of heparinin PCR. There are three physical, chemical, and enzymatic methods to remove the effect ofheparin, which we used chemical and physical methods in this experiment. First, blood sampleswere taken from a dog and poured into tubes containing EDTA and heparin. Then, ۷ vaccineswere used as a source of virus and finally, DNA extraction was performed by phenol-chloroform method.RESULTS AND DISCUSSIONTwo methods were adopted for chemical treatment. The first method was to use an ascorbicacid-oxygenated water combination. In this method, different concentrations of ascorbic acid(۲۰, ۱۰, ۵, ۱, and ۳۰ mM) and temperatures (۳۵, ۲۵, ۱۵, and ۴۵) during ultrasonic operationand ultrasonic intensity (۱۶, ۱۱, ۷, ۶, and ۲۱W / ml) during Ultrasonic surgery was considered.The second method was the use of nitrous acid at pH ۳ and ۱. ۵. For the heat treatment ofheparin, Ben Marie ۶۵ ° and ۸۵ ° for ۲ hours and ۱۲۱ ° autoclave for half, ۱, and ۲ hours wereused.CONCLUSIONAt the end of the analysis of the results in different methods of heparin inhibitory effects, itwas found that the best method among the methods performed is the use of ۸۵ ° C and ۸۵ ° Con extracted genome for ۲ hours. After that, heat treatment at ۸۵ ° C for ۲ hours on bloodsamples before extraction and finally nitrous acid (pH = ۳) treatment.

کلیدواژه ها:

Elimination of heparin inhibition ، Realtime-PCR ، Temperature ، H۲O۲/ascorbic acid ، sonication ، Nitrous acid