马驽巴贝斯虫PCR和qPCR检测方法的建立

Development of PCR and qPCR Methods for Detection of Babesia caballi

马驽巴贝斯虫是一种寄生于马属动物的血液原虫,给马产业造成巨大的经济损失。本研究旨在建立两种高效、可靠的PCR诊断技术,为进一步提高口岸检疫工作效率奠定基础。根据马驽巴贝斯虫Bc48基因序列,设计了一对特异性引物和荧光探针,建立了PCR和qPCR检测方法,测试了两种方法的特异性、灵敏性、稳定性。结果显示:建立的两种方法均具有良好的特异性;PCR方法的最低检出限为4.04×10^(2) copies/μL,qPCR方法的最低检出限为4.04×10^(1) copies/μL;对33份马血样品进行检测,结果qPCR检出的阳性样品数为23份(69.7%),而PCR检出的阳性样品数为9份(27.3%)。表明两种方法均可成功用于马驽巴贝斯虫的检验,但是荧光定量PCR方法的灵敏度明显优于PCR方法。

Babesia caballi is a parasitic protozoan in the red blood cells of equine animals and causes huge economic losses for horse industry.In this study,two effi cient and reliable PCR methods were developed to improve quarantine operations in import/export ports.A pair of specifi c primers and a probe were designed according to the sequence of Bc48 gene of B.caballi.Subsequently,specifi city,sensitivity and repeatability of the two methods were optimized for clinical use.The results showed that both methods were able to specifi cally detect B.caballi.The detection limits w ere 4.04×10^(2 )copies/μL for PCR and 4.04×10^(1) copies/μL for qPCR,respectively.Total 33 fi eld blood samples were detected these methods,in which 23(69.7%)samples were positive by qPCR and 9(27.3%)samples were positive by PCR.Overall,these two methods successfully detected B.caballi but qPCR assay was more sensitive than PCR.