粪便样本中志贺菌分离培养方法的优化

Optimization of isolation and culture method of Shigella spp.from stool samples

目的优化从腹泻患者粪便标本中分离培养志贺菌的方法。方法使用EC肉汤和志贺菌增菌液对129份粪便样本进行增菌培养,采用普通PCR方法检测ipaH基因,使用木糖赖氨酸去氧胆酸钠琼脂(XLD)培养基、麦康凯琼脂(MAC)以及沙门志贺菌培养基(SS)等选择性培养基分离培养志贺菌。使用志贺菌参考菌株,进行模拟增菌培养,利用荧光定量PCR方法分析不同增菌液的增菌效果。结果利用志贺菌增菌液在厌氧条件下增菌后的样本,ipaH基因阳性率(55.81%vs.38.76%,P<0.01)和菌株分离率(26.36%vs.37.21%,P>0.05)均高于EC肉汤。除了XLD培养基,使用MAC以及SS等选择性培养基,能够提高菌株分离率。福氏志贺菌在厌氧条件下增菌,能提高菌的含量(Ct值降低),但对宋内志贺菌的影响不明显。结论对腹泻患者粪便样本进行选择性增菌,结合分子生物学方法,使用2~3种不同选择性培养基进行分离培养,可能是提高志贺菌分离率的有效方法。

Objective To optimize the isolation method of Shigella spp.from stool samples.Methods A total of 129 stool samples were cultured with Escherichia coli broth and Shigella enrichment broth.The ipaH gene was detected by PCR.Different selective media,including Xylose Lysine Desoxycholate agar,MacConkey agar,and SS medium,were used for the isolation of Shigella spp.The reference strains of Shigella spp.were used for simulated enrichment culture.Real-time PCR was used as detection method.Results The positive rate of EC broth enrichment samples with anaerobic condition was 38.76%and 34 ipaH gene positive strains were isolated(26.36%).The positive rate of Shigella enrichment broth samples with anaerobic condition was 55.81%,which was significantly higher than that of EC broth(P<0.01),and 48 ipaH gene positive strains were isolated(37.21%).The isolation rate of Shigella spp.could be improved with other selective media such as MAC medium and SS medium except XLD medium.Anaerobic condition could increase the content of Shigella flexneri(Ct value decreased),but it had no obvious influence on Shigella sonnei.Conclusion Enrichment with 2-3 different selective culture media in combination with molecular biological method might be an effective way to improve the isolation rate of Shigella spp.in stool samples.