抗凝剂EDTA-Na_2对家禽血液指标及基因组DNA的影响

Effects of EDTA-Na_2 on Poultry Blood Indexes and Genomic DNA

目的:分析不同浓度的抗凝剂EDTA-Na2对家禽血液指标及基因组DNA抽提的影响。方法:采集鸡血,分别加入0.6(A组)、0.9(B组)、1.2(C组)、1.5(D组)、1.8(E组)和2.1(F组)mg/mL的EDTA-Na2,立即检测白细胞总数(WBC)、淋巴细胞(LYM)、中间细胞(MID)、粒细胞(GRAN)、红细胞总数(RBC)、血红蛋白(HGB)、血红蛋白含量(MCH)和血小板总数(PLT),用SPSS软件分析检测数据;用酚-氯仿法提取添加不同量抗凝剂的血液基因组DNA,并进行PCR扩增,基因组DNA和PCR产物用琼脂糖凝胶电泳检测。结果:A组样品出现肉眼可见的凝块,B组有2/7样品有凝集现象,C、D、E和F组均无凝集现象。A组凝集现象严重无法进行血液指标测定,其余5组样品中,对于RBC和HGB指标,C组与B组相比无显著性差异,与D、E、F组差异极显著(P<0.01);对于PLT,C组与B组相比无显著性差异,与D、E、F组差异显著(P<0.05);WBC无显著变化。除A组外,各组均能获得质量较好的基因组DNA,并能扩增出目的条带。结论:除A组外,不同浓度的EDTA-Na2对基因组DNA提取和PCR扩增无影响。以EDTA-Na2作为禽血抗凝剂时,建议用量应不低于1.2 mg/mL。

Objective： To analyze the effect of different concentrations of EDTA-Na2 on the poultry blood indexes and genomie DNA extraction. Methods： One milliliter blood were collected from chicken wing vein, 0.6（A）, 0.9 （B）, 1.2（C）, 1.5（D）, 1.8（E） and 2.1（F） mg/mL EDTA-Na2 anticoagulants were added to each group, respectively. Each group consisted of 7 samples. WBC, LYM, MID, GRAN, RBC, HGB, MCH and PLT were measured for each sample. The data were analyzed using SPSS. Genomic DNA was extracted with phenol-chloroform method from each sample and used for PCR to amplify [3-actin. DNA and PCR products were analyzed by agarose gel electrophoresis. Results： All samples in group A appear clots, 2/7 of group B samples appear clots, and the remaining four groups did not appear clots. We couldn＇t test blood indexes of group A because of agglutinate phenomenon. The RBC and HGB did not differ between group C and group B, but there were significant differences （P〈O.O1） when compared with group D, E, F. The PLT had the same trend（P〈0.05） as the RBC and HGB.There were no significant differences among all groups for WBC. Genomic DNA was good enough to used for PCR amplification except for group A. Conclusion： Different concentrations EDTA-Na2 have no significant effect on genomic DNA quality and PCR amplification except group A. The suggested dosage should be not less than 1.2 mg/mL when using EDTA-Na2 as anticoagulant in poultry.