沙门菌分离、筛检和确认流程技术关键点分析

doi:10.3969/j.issn.1673-8640.2020.06.003

摘要/Abstract

摘要：

目的基于标准方法构建多维组合流程,分析和评价沙门菌常规检测的技术关键点。方法构建沙门菌选择性增菌液和平板构成的检测流程,使用标准菌株检测4种选择性增菌液的回收率,验证3类5种沙门菌选择性平板的典型特征。通过实样比较不同选择性增菌液和平板组合在沙门菌检测过程中疑似菌落甄别、筛检效率和敏感性差异。结果罗伯特增菌液（RV）、四硫磺酸钠煌绿增菌液（TTB）和亚硒酸盐煌绿增菌液（SBG）回收效能与亚硒酸盐胱氨酸增菌液（SC）相差1个稀释度,前3种增菌液适合非伤寒沙门菌的增殖,SC适合伤寒和副伤寒沙门菌的增殖。双糖管筛选法符合预期参比菌株表型。除沙门菌显色平板（CAS）外,其他选择性平板的菌株验证均可能产生菌落小和硫化氢（H₂S）阴性不典型菌落;84件肉制品和水产品样品中确认阳性25件（29.76%）,检出42株沙门菌。RV、TTB、SBG、SC联合CAS和木糖赖氨酸脱氧胆酸盐平板（XLD）筛检沙门菌的敏感性分别为88%、84%、72%和0;RV和TTB、RV和SBG、TTB和SBG的组合敏感性分别为100%、92%和88%。3种增菌液与任一选择性平板组合的分离敏感性为48%~68%。双糖管筛检疑似菌落的特异性和阳性预测值较高。42株沙门菌经系统生化、质谱和血清学鉴定确认。结论经验证的双增菌液和双平板的组合能有效提高食品沙门菌检测的敏感性。梯度增菌、菌落识别和初筛效率是沙门菌检测技术的关键点。

关键词: 沙门菌, 增菌液, 选择性平板, 筛检, 技术关键点

Abstract:

Objective To analyze and evaluate the technical key points for routine detection of Salmonella,using multi-dimensional composite process based on standard method. Method Process material included Salmonella selective agars and selective enrichment broth. Recovery ratios of 4 selective enrichment broths were detected by reference isolates,and the typical colony characteristics of 5 selective agars belonging to 3 kinds were verified. Suspected colony distinguishment,screening efficiency and sensitivity were compared using different selective agars and selective enrichment broth by actual samples. Results The recovery efficiency of Rappaport Vassiliadis broth（RV）,tetrathionate broth（TTB） and selenite brilliant green broth（SBG） was one dilution different from that of selenite cystine broth（SC）. The first 3 selective enrichment broths were effective for non-typhoidal Salmonella,and SC was effective for Salmonella typhi and Salmonellaparatyphoid. Screening method of double-sugar tubes accorded with the biochemical characteristics of confirmed reference isolates. Except CHORMagar Salmonella（CAS）,the others showed atypical colony characteristics,including H₂S negative isolates and small colonies. The 25 of 84 meat samples were positive,and 42 Salmonella isolates were isolated. The sensitivities of CAS and xylose lysine deoxycholate agar（XLD） combined with RV,TTB,SBG,SC were 88%,84%,72% and 0%,respectively. The sensitivities of RV with TTB,RV with SBG,TTB with SBG were 100%,92% and 88%,respectively. The sensitivities of RV,TTB,and SBG combined with CAS or XLD were 48.00% and 68.00%,respecitvely. The method of screening Salmonella like colonies had high specificity and positive predictive value by double-sugar tubes. Totally,42 Salmonella isolates were confirmed by biochemistry identifier,mass spectrometry and serotyping. Conclusions The effective combined processes could improve the sensitivities of Salmonella detection based on verification by double selective agar and double selective enrichment broths. The technical key points include the gradient selective enrichment,distinguish of the colonies and screening of biochemistry identifier by routine detection of Salmonella.

Key words: Salmonella, Enrichment broth, Selective agar, Screening, Technical key point

中图分类号:

R446.1

刘玥, 张红芝, 顾其芳, 刘诚, 朱颖莹, 许学斌. 沙门菌分离、筛检和确认流程技术关键点分析[J]. 检验医学, 2020, 35(6): 524-530.

LIU Yue, ZHANG Hongzhi, GU Qifang, LIU Cheng, ZHU Yingying, XU Xuebin. Analysis on the technical key points of Salmonella isolation,screening and identification based on routine detection process[J]. Laboratory Medicine, 2020, 35(6): 524-530.

图/表 6

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参比菌株名称	肠道菌筛选双糖管								CAS
参比菌株名称	氧化酶	葡萄糖	甘露醇	尿酶	蔗糖	动力	硫化氢	吲哚	CAS
鼠伤寒沙门菌	-	⊕	⊕	-	-	+	+	-	酒红色
伤寒沙门菌	-	+	+	-	-	+	+	-	酒红色
甲型副伤寒沙门菌	-	+	+	-	-	+	+	+	酒红色
猪霍乱沙门菌	-	+	+	-	-	+	-	-	酒红色
猪霍乱沙门菌孔成道夫变种	-	+	+	-	-	+	+	-	酒红色
肺炎克雷伯菌	-	⊕	⊕	-	+	-	-	-	蓝色
奇异变形杆菌	-	⊕	⊕	-	+	+	-	-	淡粉色
弗劳地枸橼酸杆菌	-	⊕	⊕	-	+	+	+	-	蓝色
大肠埃希菌	-	⊕	⊕	-	+	+	-	+	蓝色

参比菌株名称	肠道菌筛选双糖管								CAS
参比菌株名称	氧化酶	葡萄糖	甘露醇	尿酶	蔗糖	动力	硫化氢	吲哚	CAS
鼠伤寒沙门菌	-	⊕	⊕	-	-	+	+	-	酒红色
伤寒沙门菌	-	+	+	-	-	+	+	-	酒红色
甲型副伤寒沙门菌	-	+	+	-	-	+	+	+	酒红色
猪霍乱沙门菌	-	+	+	-	-	+	-	-	酒红色
猪霍乱沙门菌孔成道夫变种	-	+	+	-	-	+	+	-	酒红色
肺炎克雷伯菌	-	⊕	⊕	-	+	-	-	-	蓝色
奇异变形杆菌	-	⊕	⊕	-	+	+	-	-	淡粉色
弗劳地枸橼酸杆菌	-	⊕	⊕	-	+	+	+	-	蓝色
大肠埃希菌	-	⊕	⊕	-	+	+	-	+	蓝色

沙门菌型	SC		TTB		RV		SBG
沙门菌型	靶菌	加标	靶菌	加标	靶菌	加标	靶菌	加标
伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
甲型副伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
鼠伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-10	10^-9	10^-10	10^-9
猪霍乱沙门菌	10^-7	10^-6	10^-10	10^-8	10^-10	10^-8	10^-9	10^-8
猪霍乱沙门菌孔成道夫变种	10^-7	10^-6	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8

沙门菌型	SC		TTB		RV		SBG
沙门菌型	靶菌	加标	靶菌	加标	靶菌	加标	靶菌	加标
伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
甲型副伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
鼠伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-10	10^-9	10^-10	10^-9
猪霍乱沙门菌	10^-7	10^-6	10^-10	10^-8	10^-10	10^-8	10^-9	10^-8
猪霍乱沙门菌孔成道夫变种	10^-7	10^-6	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8