聚合酶链反应-酶联免疫法筛选孢子丝菌特异性探针

Screening of species-specific probes for Sporothrix schenckii by PCR-ELISA

目的筛选出对孢子丝菌结合效率相对较高的探针。方法以50株纯培养的孢子丝菌及标准株为样本,以毛霉、烟曲霉、念珠菌各1株为阴性对照,用聚合酶链反应-酶联免疫法(PCR-ELISA)对已报道的5个孢子丝菌特异性探针(U26852、U26866、U26866'、M85053及AF117945)进行筛选。记录各探针与合成DNA片段杂交底物显色与否及酶免疫测定指数(EI)值。结果 PCR产物测序证实5个探针均位于产物序列上;在相同的ELISA条件下,探针U26852显色最强,EI值最高。结论针对28Sr RNA的探针U26852是目前与孢子丝菌结合效率相对较高的探针。

ObjectiveTo screen out the highest efficient probe from those that have been reported forSporothrix schenckii （S.schenckii）.MethodsFifty strains of culturedS. schenckii and standard strain were used as experimental samples, and 1 strain of Mucor, Aspergillus funigatus, and Monilia albican were used as negative controls. By using the PCR-ELISA format, the most speciifc and sensitive probe was selected from ifve speciifc probes forS. shenckii that have been reported so far, namely probe U26852, probe U26866, probe U26866′, probe M85053 and probe AF117945. The sensitivity and speciifcity of the probes were determined based on the color and the optical density.ResultsThe experiment conifrmed that 5 probes were all located on the sequences of the product by sequencing of the PCR products. Under the same hybridization and PCR-ELISA conditions, all the 50 isolates ofS. schenckii had a stronger colouration and higher value of EI to probe U26852 than to the other 4 probes.ConclusionThe probe U26852 （targeting the 28SrRNA gene） forS. schenckii is highly efifcient.