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目的建立犬贾第虫巢式PCR检测方法并进行验证。方法根据犬贾第虫Rnase P基因序列设计两对特异性引物,通过优化Mg2+浓度和退火温度建立巢式PCR检测方法,并进行灵敏度和特异性验证。用优化的巢式PCR方法检测60份犬临床粪样。结果建立的巢式PCR方法的最佳Mg2+浓度为3.0 mmol/L;最佳退火温度为53℃;对犬贾第虫的最低检测量可达1个虫体DNA/ml;对柔嫩艾美尔球虫、伊氏锥虫、弓形虫、阴道毛滴虫、犬心丝虫等寄生虫基因组DNA的扩增结果均为阴性;60份犬临床粪样贾第虫的检出率为61.7%。结论建立的巢式PCR检测方法具有较高的灵敏度和特异性,可用于犬贾第虫感染的临床检测。
Objective To establish and validate nested PCR detection method of Giardia lamblia. Methods Two pairs of primers were designed according to the sequence of Rnase P gene of canine, and the nested PCR method was established by optimizing the concentration of Mg2 + and the annealing temperature. The sensitivity and specificity of the method were verified. Sixty canine clinical fecal samples were tested using the optimized nested PCR method. Results The optimum concentration of Mg2 + in nested PCR was 3.0 mmol / L. The optimal annealing temperature was 53 ℃. The detection limit of Giardia was 1 worm DNA / ml. Coccidia, Trypanosoma toxoplasma, Toxoplasma gondii, Trichomonas vaginalis, canine heartworm and other parasite genomic DNA amplification results were negative; 60 cases of dog clinical fecal Giardia detection rate was 61.7%. Conclusion The established nested PCR detection method has high sensitivity and specificity and can be used for the clinical detection of Giardia canine infection.