气单胞菌aerA和hlyA两基因在溺死诊断中的应用

目的建立溺死尸体脏器气单胞菌气溶素(aerA)和溶血素(hlyA)两基因的PCR-CE检测方法,验证该方法的特异性和灵敏度,并探讨其在溺死诊断中的应用价值。方法设计气单胞菌(Aeromonas)特异性引物aer-A1和hlyA-1,构建PCR-CE方法;扩增52种标准株DNA;确定最低DNA检测浓度;检测16只实验猪和40例真实案件尸体组织样本,分别计算两对引物总阳性率。结果引物aerA-1可特异性扩增杀鲑气单胞菌和温和气单胞菌,产物片段为180bp,灵敏度分别为0.034、0.92ng;引物hlyA-1可特异性扩增杀鲑气单胞菌和嗜水气单胞菌,产物片段为150bp,灵敏度分别为0.94、0.034ng。利用aerA-1检测溺死实验猪和真实案件尸体组织样本,溺死阳性率分别为70.00%、78.38%;利用hlyA-1检测溺死实验猪和真实案件尸体组织样本,溺死阳性率分别为80.00%、83.78%。结论基于气单胞菌aerA基因和hlyA基因建立的PCR-CE检测方法进行溺死诊断,快速灵敏,特异性好,有良好的应用前景。

Applicability of Aeromonas Genes of aerA and hlyA into Diagnosis of Drowning

Objective To establish a PCR-CE method for detection of Aeromonas genes,aerolysin(aerA)and hemolysin(hlyA),from the tissues of drowning cadavers along with the specificity and sensitivity being tested for the evaluation of its forensic applicability into drowning diagnosis.Methods Targeted to the conserved sequences of Aeromonas,the specific primers(aerA-1 and hlyA-1)were designed for amplification of the purposed genetic fragments.52 species were respectively tested to verify the specificity of the amplified fragments,with the sensitivity being measured through the DNA of positive control samples.The positive rate of aerA-1 and hlyA-1 was calculated from detection into the 16 experimental pigs and 40 cadavers.Results Primer aerA-1 was able to specifically amplify Aeromonas salmonicida and Aeromonas veronii,resulting in its PCR products of 180bp and the sensitivity of 0.034ng and 0.92ng for the two bacteria,respectively.Likewise,primer hlyA-1 amplified Aeromonas salmonicida and Aeromonas hydrophila specifically,having its PCR products of 150bp and the sensitivity of 0.94ng and 0.034ng for the related two bacteria.The aerA-1 showed its positive rate of individual 70.00%and 78.38%in detecting the drowning experimental pigs and cadavers;so did the primer hlyA-1 of 80.00%and 83.78%,respectively.Conclusion Through detection of Aeromonas genes of aerA and hlyA,the established PCR-CE approach is eligible for drowning diagnosis,demonstrating its fast,sensitive and specific prominence,having good applicative prospect for forensic utilization.