CN110551792A - 一种快速v-p试验法以及用于该方法的试剂盒 - Google Patents
一种快速v-p试验法以及用于该方法的试剂盒 Download PDFInfo
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Abstract
本发明公开了一种快速V‑P试验法以及用于该方法的试剂盒。所述的快速V‑P试验法,包括以下步骤:(1)将试验菌种接种于改良MR‑VP肉汤培养基中,37℃保温3‑4h,哈夫尼亚菌在22‑25℃保温;(2)保温结束后往培养基中依次加入A液2滴,B液3滴,C液2滴,充分混合2‑5min,呈现红色判定为阳性;若无红色出现,置于室温或37℃恒温箱,15min内仍不显现红色、判定为阴性。其中,A液为0.5%w/w肌酸溶液;B液为含5%w/wα‑萘酚(1‑naphthol)的95%(w/w)酒精溶液;C液为40%w/v氢氧化钾水溶液。该方法加快了出现阳性反应的速度,3‑4h即可判读结果。增强显色效果,鲜明直观。可同时适用于肠杆菌科和微球菌科细菌的检测,没有对待检菌落的特殊要求。培养基、试剂用量少,价格低廉;配方简单,性能稳定。
Description
技术领域
本发明涉及一种V-P试验法,还涉及用于该方法的试剂盒。本发明属于微生物检测技术领域。
背景技术
肠杆菌科和微球菌科细菌属种繁多、分布广泛、寄主范围大,有几十个与医学有关的菌属,占临床分离菌总数的50%,将近50%的败血症、70%以上的泌尿道感染是由此类菌引起的,另外由其引起的食物中毒事件已成为世界性的公共卫生问题,是人类健康的潜在杀手,如何避开设备条件的限制,利用快速简便的生化试验实现对肠杆菌科和微球菌科细菌的属种快速鉴别,是我们需要研究攻破的重点课题。
V-P试验,又名二乙酰试验。其原理是某些细菌在糖代谢过程中,分解葡萄糖产生丙酮酸,丙酮酸脱羧产生乙酰甲基甲醇,乙酰甲基甲醇在碱性环境中,被空气中的氧氧化为二乙酰,进而与培养基内蛋白胨中精氨酸所含的胍基起作用,生成红色化合物,则为V-P试验阳性。V-P试验可以用于以下用途:
①肠杆菌属与埃希菌属,葡萄球菌属与微球菌属的属间鉴别;
②肺炎克雷伯菌、产酸克雷伯菌、臭鼻克雷伯菌、鼻硬结克雷伯菌间的菌种鉴别;
③蜂房哈夫尼亚菌与小肠结肠炎耶尔森菌及单核细胞增生李斯特菌的菌种鉴定。
国内外常用的V-P试验法有:O’Meara氏法、Barritt氏法、Coblentz氏法等,其中最快要24h,慢的要4天才能得出检测结果;在检测芽胞杆菌和葡萄球菌等其它细菌时,通用培养基中的磷酸盐会阻碍乙酰甲基甲醇的产生,造成假阴性结果;另外有些方法仅能检测产酸的培养物。不但检测时间长,试剂不稳定,适用范围也受到限制。
鉴于此种现状,本发明建立了一种快速V-P试验法,在保证结果可靠性的同时解决了以往方法存在的问题和不足。快速V-P试验法的先进性在于:1、加快了出现阳性反应的速度,3-4h即可判读结果。2、增强显色效果,鲜明直观。3、同时适用于肠杆菌科和微球菌科细菌的检测,没有对待检菌落的特殊要求。4、培养基、试剂用量少,价格低廉;配方简单,性能稳定。
发明内容
本发明的目的在于建立一种快速V-P试验法以及用于该方法的试剂盒。
为了达到上述目的,本发明采用了以下技术手段:
本发明的一种快速V-P试验法,包括以下步骤:
(1)将试验菌种接种于改良MR-VP肉汤培养基中,37℃保温3-4h,哈夫尼亚菌在22-25℃保温;
所述的改良MR-VP肉汤通过以下方法制备:酵母膏1g、植物胨5g、多胨7g、氯化钠5g、葡萄糖10g、琼脂5g,加蒸馏水1000ml,调节pH7.0,121℃15min灭菌,试管分装;
(2)保温结束后往培养基中依次加入A液2滴,B液3滴,C液2滴,充分混合2-5min,呈现红色判定为阳性;若无红色出现,置于室温或37℃恒温箱,15min内仍不显现红色、判定为阴性;
其中,A液为0.5%w/w肌酸溶液;B液为含5%w/wα-萘酚(1-naphthol)的95%w/w酒精溶液;C液为40%w/v氢氧化钾水溶液。
其中,优选的,所述的快速V-P试验法可用于肠杆菌属与埃希菌属,葡萄球菌属与微球菌属的属间鉴别;或肺炎克雷伯菌、产酸克雷伯菌、臭鼻克雷伯菌、鼻硬结克雷伯菌间的菌种鉴别;或蜂房哈夫尼亚菌与小肠结肠炎耶尔森菌及单核细胞增生李斯特菌的菌种鉴定。
进一步的,本发明还提出了一种用于快速V-P试验法的试剂盒,所述的试剂盒包括:
改良MR-VP肉汤:酵母膏1g、植物胨5g、多胨7g、氯化钠5g、葡萄糖10g、琼脂5g、蒸馏水1000ml,pH7.0,121℃15min灭菌,按照0.2ml每支分装;
A液:0.5%w/w肌酸溶液;
B液:含5%w/wα-萘酚(1-naphthol)的95%w/w酒精溶液;
C液:40%w/v氢氧化钾水溶液。
更进一步的,本发明还提出了所述的试剂盒在快速V-P试验法检测中的应用。
快速V-P试验法以检查某些细菌在葡萄糖代谢中是否产生乙酰甲基甲醇为鉴定依据。乙酰甲基甲醇在碱性环境中可被空气中的氧氧化为丁二酮,丁二酮与蛋白胨中精氨酸所含的胍基作用生成红色化合物,即为V-P试验阳性。本发明所采用的关键技术手段包括:
1、胍基部分的游离NH2为产生颜色所需要的成分。肌酸或肌酸酐可供给比精氨酸更多的胍基,有加快V-P试验出现阳性反应的作用。
2、加入α-萘酚可使V-P试验更为灵敏,提高V-P试验灵敏度约50倍而不降低试验的特异性,其酒精溶液还有增强颜色的作用。
3、氢氧化钾作为氧化剂可加速乙酰甲基甲醇氧化为丁二酮,并有助于二氧化碳的吸收。
4、在试验中首先加入α-萘酚使其先与丁二酮的胍基复合物结合,然后加入40%氢氧化钾溶液。若加入顺序颠倒氢氧化钾可与蛋白胨某些成分反应产生橙红色而造成假阳性。
5、用氯化钠代替通用培养基中的磷酸盐可以避免在检测芽胞杆菌和葡萄球菌等其它细菌时,培养基中的磷酸盐阻碍乙酰甲基醇的产生,造成假阴性结果。从而真正实现了直观、快速、简便、准确、经济、适用。
相较于现有技术,本发明的有益效果是:
1、加快了出现阳性反应的速度,3-4h即可判读结果。
2、增强显色效果,鲜明直观。
3、同时适用于肠杆菌科和微球菌科细菌的检测,没有对待检菌落的特殊要求。
4、培养基、试剂用量少,价格低廉;配方简单,性能稳定。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1快速V-P试验法试剂盒的制备
包括:
改良MR-VP肉汤培养基:酵母膏1g、植物胨5g、多胨7g、氯化钠5g、葡萄糖10g、琼脂5g、蒸馏水1000ml,pH7.0,121℃15min灭菌,按照0.2ml每支试管分装。
A液:0.5%w/w肌酸溶液;
B液:含5%w/wα-萘酚(1-naphthol)的95%w/w酒精溶液;
C液:40%w/v氢氧化钾水溶液。
实施例2快速V-P试验法的建立
1、快速V-P试验法的建立
步骤:从平板挑取一颗菌落接种于0.2mL改良MR-VP肉汤培养基(实施例1制备)中,37℃保温3-4h(哈夫尼亚菌在22-25℃保温)。
结果观察:往试管中依次加入A液2滴,B液3滴,C液2滴,A液,B液,C液按照实施例1制备,充分混合2-5min,呈现红色判定为阳性;若无红色出现,置于室温或37℃恒温箱,15min内仍不显现红色、判定为阴性。
用于质量控制的阳性菌株:肺炎克雷伯氏菌(46114-8)、金黄色葡萄球菌(ATCC6538);阴性菌株:大肠埃希菌(8099)、藤黄微球菌。
2、快速V-P试验的准确性观察
同时以常规法(O’Meara氏法)作对照。具体操作方法为:将试验菌接种于通用培养基(蛋白胨0.5g、葡萄糖0.5g、磷酸氢二钾0.5g以及水100ml),于36℃±1℃培养48h,培养液1mL加O‘Meara试剂(加有0.3%肌酸的40%氢氧化钠水溶液)1mL,摇动试管1min~2min,静置于室温或36℃±1℃恒温箱,若4h内不呈现伊红即判定为阴性。
通过对标准菌株和经API 20E、API Staph鉴定的从临床、环境、食品标本中分离到的154株菌(其中包括肠杆菌科的肠杆菌属、埃希菌属、志贺氏菌属、柠檬酸杆菌属、哈夫尼亚菌属、摩根菌属、克雷伯氏菌属、普罗威登斯菌属、沙门氏菌属、沙雷氏菌属、耶尔森氏菌属和微球菌科的金黄色葡萄球菌属、微球菌属,单核细胞增生李斯特菌,共14个菌属,27个菌种的细菌)的接种试验表明,使用本发明的快速V-P试验法检测生成红色化合物的敏感性为97.67%,特异性为100.00%,约登指数为97.67%,检测时间为3-4h,证明该快速V-P试验法的准确性很高,且快速。快速V-P试验法与O’Meara氏法的检测结果如表1所示:
表1
3、快速V-P试验法与O’Meara氏法检测结果的统计学分析比较
将两种方法同时对154株菌的试验结果进行统计学分析比较,证明两种检测方法无显著差异(X2=0.05,p>0.05)。
4、快速V-P试验用试剂的稳定性观察
将质控合格的试剂置4-8℃条件下保存。每月同新配制的试剂一同用标准菌株进行验证,结果一致,连续12个月性能稳定。
Claims (4)
1.一种快速V-P试验法,其特征在于,包括以下步骤:
(1)将试验菌种接种于改良MR-VP肉汤培养基中,37℃保温3-4h,哈夫尼亚菌在22-25℃保温;
所述的改良MR-VP肉汤通过以下方法制备:酵母膏1g、植物胨5g、多胨7g、氯化钠5g、葡萄糖10g、琼脂5g,加蒸馏水1000ml,调节pH7.0,121℃15min灭菌,试管分装;
(2)保温结束后往培养基中依次加入A液2滴,B液3滴,C液2滴,充分混合2-5min,呈现红色判定为阳性;若无红色出现,置于室温或37℃恒温箱,15min内仍不显现红色、判定为阴性;
其中,A液为0.5%w/w肌酸溶液;B液为含5%w/wα-萘酚(1-naphthol)的95%w/w酒精溶液;C液为40%w/v氢氧化钾水溶液。
2.如权利要求1所述的快速V-P试验法,其特征在于,所述的快速V-P试验法可用于肠杆菌属与埃希菌属,葡萄球菌属与微球菌属的属间鉴别;或肺炎克雷伯菌、产酸克雷伯菌、臭鼻克雷伯菌、鼻硬结克雷伯菌间的菌种鉴别;或蜂房哈夫尼亚菌与小肠结肠炎耶尔森菌及单核细胞增生李斯特菌的菌种鉴定。
3.一种用于快速V-P试验法的试剂盒,其特征在于,所述的试剂盒包括:
改良MR-VP肉汤:酵母膏1g、植物胨5g、多胨7g、氯化钠5g、葡萄糖10g、琼脂5g、蒸馏水1000ml,pH7.0,121℃15min灭菌,按照0.2ml每支分装;
A液:0.5%w/w肌酸溶液;
B液:含5%w/wα-萘酚(1-naphthol)的95%w/w酒精溶液;
C液:40%w/v氢氧化钾水溶液。
4.权利要求3所述的试剂盒在快速V-P试验法检测中的应用。
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