PCR和Southern Blot检测土拉弗氏菌气溶胶

PCR and Southern Blot Based Detection of Francisella tularensis Aerosols

为提高检测土拉弗氏菌的特异性和敏感性，建立了土拉菌ＰＣＲ及核酸杂交检测方法。运用平板计数、多聚酶链反应对土拉菌气溶胶稳定性进行了比较，结果表明ＰＣＲ具有较高灵敏度，并且在采样后３小时ＰＣＲ就可以得出定性结果，而平板计数则需要３～７天。采用ＰＣＲ法合成了土拉菌３７６－ｂｐ探针，分别对细菌菌液、５６８－ｂｐＰＣＲ产物和气溶胶样品进行杂交，结果表明菌悬液直接杂交可检出１０５ＣＦＵ左右的细菌，检测ＰＣＲ产物可达４０ｐｇ。

For the specific and sensitive detection of bioaerosols,four primers (3pairs) were designed to detect F.tularensis by the polymerase chain reaction,based on the previously published nucleotide sequence of T cell epitopes of a F.tularensis membrane protein.Amplification of purified F.tularensis chromosomal DNA with the 3 pairs of primers resulted in 3 different products with sizes consistent with those predicted from sequence data (211-bp,347-bp and 568-bp).The stability of Francisella tularensis aerosol was aslo studied by use of PCR and plate counting separately.Results showed that PCR is a more sensitive tool for the detection of bioaerosls within 3 hours,while traditional plate count need 3 or more days.Therefore,the method would provide a more reliable estimat of airborne bacterial concentrations compared to traditional plate counts.Probes for the detection of Francisella tularensis were senthesized by PCR.Nucleic acid hybridization results showed that 10 5CFU of Francisella tularensis could be detected by direct hybridization,whileas the PCR product,the detection limit was 40pg.PCR is a helpful tool for the detection of F.tularensis,which should enable the rapid confimation of clinical diagnosis of tularemia.