通用引物PCR方法快速检测兔眼真菌性角膜溃疡

Detection of fungal corneal ulcer in rabbit eyes by general primer-mediatied polymerase chain reaction

目的 应用真菌通用引物聚合酶链反应PCR ,对兔眼真菌性角膜溃疡进行快速检测。方法 建立兔眼烟曲霉菌、白色念珠菌和镰刀菌真菌性角膜溃疡的模型 ,根据国外文献报道的基因序列选出真菌通用引物 ,一对寡核苷酸引物B2F(5’ ACTTTCGTAG GATAG 3’)和引物B4R(5’ TGATCGTCTTCGATAAATA 3’)进行聚合酶链反应。结果 在 6 87bp处出现DNA扩增带者为阳性。动物造型 3d后 ,通过PCR技术、常规刮片和培养 3种实验室手段诊断为真菌性角膜炎的敏感性分别为 90 .0 %、33.3%和 4 0 .0 % ;5d后 ,PCR技术、常规刮片和培养的敏感性分别为 96 .6 %、5 0 .0 %和 6 0 .0 % .结论 通用引物PCR技术检测兔眼真菌性角膜溃疡快速、敏感、可靠。

Objective To detect fungal corneal ulcer in rabbit eyes by general primer mediatied polymerase chain reaction(GP PCR).Methods We set up the fungal corneal ulcer model of aspergillus fumigatus,candida albicans,fusarium in rabbit eyes;According to fungal gene orders that foreign literatures reported, we devised and chosed out a pair of oligonucleotide primers B2F(5' ACTTTCGTAGGATAG 3')and B4R(5' TGATCGTCTTCGATAAATA 3'),whose expanded fraction appeared at 687bp.Results A 687bp specific DNA product was successfully amplified.In model of mycotic keratitis, the sensitivity of PCR, scrapes and culture were 90.0% , 33.3% and 40.0% at the 3rd day;The sensitivity of PCR, scrapes and culture were 96.6%,50.0% and 60.0% at the 5th day.Conclusion The results indicate that GP PCR is arapid,sensitive,and reliable detection method for fungal corneal ulcer in rabbit eyes.