PCR毛细管电泳法检测嗜水气单胞菌gyrB和16SrRNA基因：溺死诊断方法

Diagnosis of drowning by detecting gyr B and 16S rRNA genes of Aeromonas hydrophila using PCR-capillary electrophoresis

目的建立嗜水气单胞菌gyr B和16SrRNA基因PCR毛细管电泳检测方法,探讨它们在淡水溺死诊断中的应用价值。方法提取人、18种浮游生物(白色念珠菌、嗜水气单胞菌及16种藻类),以及经微波消解-真空抽滤-电镜扫描法检测过的30例尸体(其中生前淡水溺死28例,陆地自然死亡2例)的组织样本的DNA(肺、肝、肾各1份/例),分别使用引物AH(gyr B基因)及引物Ah(16SrRNA基因)进行扩增,扩增产物用毛细管电泳法检测。结果人、白色念珠菌、16种藻类DNA扩增后毛细管电泳检测结果均为阴性,而嗜水气单胞菌结果均为阳性,其产物大小分别为195 bp,350 bp。分别利用引物AH和Ah对28例淡水溺死尸体样本肺、肝、肾进行PCR毛细管法进行检测,嗜水气单胞菌的检出率:AH分别为96.4%,71.4%,60.7%;Ah分别为75.0%,42.9%,32.1%,计算得到溺死者的体循环脏器中嗜水气单胞菌检验的总阳性率分别为82.1%,53.6%,2例陆地自然死亡案件尸体样本检测结果均为阴性。两对引物AH与Ah对嗜水气单胞菌检出率有显著性差异(P<0.05)。结论 PCR毛细管电泳法检测嗜水气单胞菌gyr B基因用于诊断淡水溺死灵敏度较高,可作为诊断的辅助性方法;联用16SrRNA基因可提高该菌的检出率,增强MDVF-Auto SEM法诊断淡水溺死的证据力。

Objective To establish a method for diagnosis of freshwater drowning by amplifying gyr B and 16 S rRNA genes of Aeromonas hydrophila using PCR coupled with capillary electrophoresis（CE）. Methods DNA samples were extracted from human, 18 planktons（including Candida albicans, Aeromonas hydrophila, and 16 species of algae）, and 30 cases of tissue samples（including the lung, liver, and kidney, all examined with microwave digestion-vacuum filtration-automated scanning electron microscopy） from human cadavers, including 28 freshwater drowning victims and 2 with natural death. The DNA samples were amplified with the primer AH（for gyr B gene） and primer Ah（for 16 S rRNA gene）, and the products were analyzed with CE. Results PCR amplification followed by CE yielded negative results for DNA of human, Candida albicans and16 species of algae, whereas a positive result was found for Aeromonas hydrophila DNA with PCR products of 195 bp（with primer AH） and 350 bp（with primer Ah）. In the 28 drowning cases, the detection rates of Aeromonas hydrophila using primer AH were 96.4% in the lung tissue, 71.4% in the liver tissue, and 60.7% in the kidney, as compared with the rates of 75.0%,42.9%, and 32.1% using primer Ah, respectively. The positive rates for Aeromonas hydrophila in the organs of the drowning victims were 82.1% and 53.6% with primer AH and primer Ah, respectively. The detection showed negative results in the 2cases of natural deaths. The two primers produced significantly different detection rates of Aeromonas hydrophila（P〈0.05）.Conclusion PCR coupled with CE for detecting gyr B gene of Aeromonas hydrophila has a high sensitivity in assisting a diagnosis of freshwater drowning. Detection of both the gyr B gene and 16 S rRNA gene of Aeromonas hydrophila can yield more convincing evidence of the diagnosis of freshwater drowning.