5种常见念珠菌反向斑点杂交技术的研究

The establishment of five reverse cross blot hybridization techniques for diagnosis of candida species

目的建立白色念珠菌、光滑念珠菌、克柔念珠菌、近平滑念珠菌、热带念珠菌的反向斑点杂交诊断技术。方法针对念珠菌ERG11保守基因设计引物和杂交探针,对探针进行标记然后固定在尼龙膜形成一种杂交膜,将PCR产物点样在杂交膜进行斑点杂交,显色。结果通过通用引物扩增五种常见念珠菌DNA片段,大小为350bp。构建了5种念珠菌的反向斑点杂交膜,与其PCR产物反应特异性好,灵敏度高。结论本研究建立的反向斑点杂交技术可快速、准确地鉴定临床常见念珠菌,有望在临床微生物诊断中广泛应用。

Aim To establish five PCR-based reverse cross blot hybridization techniques for diagnosis of candida species.Methods The primer and specific oligonucleotide probes to conservative ERG11 gene of five common candida species were designed based on the gene fragments of PCR amplified products and hybond membrane was binded with the labeled probe for dot blot hybridization and coloration.Results The size of PCR amplified DNA fragment of five candida species was 350bp.The specificity and sensibility of hybond membrane reacted with PCR products of five common candida species were high.Conclusion The established PCR-based reverse cross blot hybridization methods allow exactly identify clinical common Candida species and wide application in clinical microorganism diagnosis.