CN110286010A - 一种直肠内液样本专用前处理液及其制备方法 - Google Patents
一种直肠内液样本专用前处理液及其制备方法 Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
- A01N43/80—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,2
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/12—Iodine, e.g. iodophors; Compounds thereof
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- General Physics & Mathematics (AREA)
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Abstract
本发明涉及一种具有霉菌抑制和细胞破碎功能的直肠内液样本专用前处理液,包含的组分为甲基异噻唑啉酮、十二烷基硫酸钠、乙二胺四乙酸二钠、异丁醇、壳聚糖‑碘络合物和磷酸盐缓冲液。本发明还涉及上述前处理液的制备方法。本发明的前处理液可以向样本提供稳定的环境,因为甲基异噻唑啉酮与壳碘的联合应用,对直肠内液样本检测前处理过程中的微生物尤其是霉菌具有消杀和抑制滋生的作用,同时十二烷基硫酸钠、异丁醇及蒸馏水对有型细胞的三重破碎功能叠加,使样本内的有型细胞破碎释放出细胞内物质,防止样本内有形物质凝固和沉淀,保证样本检测的准确性。
Description
技术领域
本发明属于医药用品领域,特别涉及一种直肠内液样本专用前处理液及其制备方法。
背景技术
直肠内液是一种医学临床检验使用的一种人体样本,可用于钾、钠、氯、PH值、血红素蛋白等生物化学分析和微生物检测。直肠内液内含肠内消化道分泌液、病理性渗出液、残留食靡、有型细胞(红细胞,白细胞,植物、动物、微生物细胞等),可能还有残留的粪便尤其是稀便等肠内容物。由于直肠内环境的特殊性,其中细菌、霉菌、寄生虫等存在较多。前处理液是体外诊断试剂的组成部分,需要一定的有效期,一般为1~2年。液基试剂保管过程中往往有霉菌生长,致使试剂内出现霉菌菌丝和孢子等形成的丝状物、絮状物甚至块状物。霉菌生长将严重影响检测结果的准确性。为解决以上问题,需要一种为样本提供稳定的环境,具有防腐灭菌尤其是抑制霉菌功效,并能使细胞破裂,防止样本内有形物质凝固和沉淀,清除样本中的粘液以降低黏度,提高分析检测的精准度和可靠性的预处理技术。
发明内容
本发明的第一目的是为解决现有技术中存在的问题,提供了一种直肠内液样本专用前处理液,该前处理液能快速地处理不同来源的直肠内液样本,保存过程中抑制霉菌污染,破碎各种有型细胞,从而实施直肠内液的各项化验项目,提高检测的精准度。
本发明的第二目的是提供上述直肠内液样本专用前处理液的制备方法。
本发明通过以下技术方案来实现:
一、一种直肠内液样本专用前处理液,每升溶液中包含的组分及含量为:甲基异噻唑啉酮0.05~1.5ml、十二烷基硫酸钠0.2~0.6g、乙二胺四乙酸二钠1.6~3.2g、异丁醇3~7ml、壳聚糖-碘络合物55~80g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
具体的,每升溶液中包含的组分及含量为:甲基异噻唑啉酮0.1ml、十二烷基硫酸钠0.4g、乙二胺四乙酸二钠2g、异丁醇4ml、壳聚糖-碘络合物65g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
具体的,每升溶液中包含的组分及含量为:甲基异噻唑啉酮0.05ml、十二烷基硫酸钠0.2g、乙二胺四乙酸二钠1.6g、异丁醇3ml、壳聚糖-碘络合物55g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
具体的,每升溶液中包含的组分及含量为:甲基异噻唑啉酮1.5ml、十二烷基硫酸钠0.6g、乙二胺四乙酸二钠3.2g、异丁醇7ml、壳聚糖-碘络合物80g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
具体的,该前处理液的基质为蒸馏水。
二、上述的直肠内液样本专用前处理液的制备方法,具体步骤如下:
步骤一、磷酸缓冲液的制备:无水磷酸氢二钠加入于蒸馏水中,搅拌至完全溶解,再将无水磷酸二氢钠溶于上液中,定容;
步骤二、将乙二胺四乙酸二钠、十二烷基硫酸钠加入到步骤一得到的磷酸盐缓冲液中,在57-60℃水浴中加热溶解;
步骤三、将异丁醇加入步骤二制得的溶液中,充分搅拌使之完全溶解;
步骤四、将壳碘、甲基异噻唑啉酮加入步骤三制得的溶液中,搅拌使之混匀。
本发明的机理和及各化工原料在本发明中的作用效果如下:
甲基异噻唑啉酮(MIT)是一种广谱的杀菌防腐剂,能有效杀灭藻类、细菌和真菌。该活性单剂有效用量少,无毒无污染,极易混合在各类配方中,PH使用范围广,稀释使用浓度后,很容易被生物降解为无毒无污染物质。毒性低,排放无残留,与各种乳化剂、表面活性剂及蛋白质成份配伍性好。应用于本专利配伍,是唯一对氧化-还原反应无干扰作用的试剂防腐剂。
壳碘(壳聚糖-碘络合物)兼具壳聚糖和碘的性能。壳聚糖在体内能趋化白细胞,诱导局部巨噬细胞,增强其吞噬功能和水解活性,刺激其产生淋巴因子和炎性介质,从而增强机体的抗感染能力,促进伤口的愈合,具有免疫调节、抑菌、抗肿瘤等特性。壳聚糖分子H支链和碘分子的H链的结合,且物质正负电相吸,从而碘分子被壳聚糖分子包封成纳米级囊和微囊的形式。作用时,纳米级碘分子从壳聚糖分子中持续缓释出来杀菌消炎,使碘能保持持续72小时的抗菌消炎作用,具有广谱抗微生物效能,对霉菌具有特殊功效。纳米级生物萃取的壳碘因子与临床外用的化学合成碘不同:后者是无机碘,是强氧化剂,常用于体表皮肤粘膜消毒,只有短时间的消杀作用。前者是生物活性碘,和人体自体抗炎细胞的分子结构相似,具备高效的抗菌消炎功效。
十二烷基硫酸钠是一种阴离子表面活性剂,具有细胞破碎功能。它还是一种离子对试剂,强亲水离子反作用于样品分子的中性离子对,用于同时分离带电分子和非带电分子。使试剂分析结果具有极好的重现性,增加色染保留时间,改善分离性质,显著缩短分离时间,防止样本内有形物质凝固和沉淀。
乙二胺四乙酸二钠是一种络合剂,主要用于络合亚铁离子,控制聚合反应速度。利用络合剂的分散、悬浮功能,阻止水中沉淀物的产生。络合剂与Fe2+形成的配合物可吸收部分可见光,生成强氧化型的自由基,使反应初期Fe2+快速氧化为Fe3+,快的氧化速度利于生成低晶化度的γ-羟基氧化铁(γ-FeOOH),亦防止样本内有形物质凝固和沉淀,并具有清除样本中的粘液降低黏度的功能。
异丁醇是常用有机溶剂及萃取剂,防止样本内有形物质凝固和沉淀,且具有细胞破碎功能,还可以减少样本中可能存在的蛋白质、糖、甘油三脂、胆固醇和纤维蛋白原等物质的干扰,提高检测结果的准确性。
采用上述技术方案的积极效果:提供一种具有具有霉菌抑制和细胞破碎功能直肠内液样本前处理液,该前处理液可以向样本提供稳定的环境,因为甲基异噻唑啉酮与壳碘的联合应用,对直肠内液样本检测前处理过程中的微生物尤其是霉菌具有消杀和抑制滋生的作用,同时十二烷基硫酸钠、异丁醇及蒸馏水对有型细胞的三重破碎功能叠加,使样本内的有型细胞破碎释放出细胞内物质,防止样本内有形物质凝固和沉淀,保证样本检测的准确性。
具体实施方式
下面结合具体实施例和试验例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与改型,均落在本发明的保护范围内。
实施例1
实施例1说明本发明高浓度前处理液
1、制备0.5mol/L、pH7.4磷酸盐缓冲液1000ml;
2、将乙二胺四乙酸二钠3.2g、十二烷基硫酸钠0.6g加入到步骤1得到的磷酸盐缓冲液中,在57℃水浴中加热溶解;
3、将异丁醇7ml加入步骤2制得的溶液中,充分搅拌使之完全溶解;
4、将壳碘80g、甲基异噻唑啉酮1.5ml加入步骤3制得的溶液中,搅拌使之混匀。
实施例2
实施例2说明本发明优选前处理液
1、制备0.5mol/L、pH7.4磷酸盐缓冲液1000ml;
2、将乙二胺四乙酸二钠2.0g、十二烷基硫酸钠0.4g加入到步骤1得到的磷酸盐缓冲液中,在60℃水浴中加热溶解;
3、将异丁醇4ml加入步骤2制得的溶液中,充分搅拌使之完全溶解;
4、将壳碘65g、甲基异噻唑啉酮0.1ml加入步骤3制得的溶液中,搅拌使之混匀。
实施例3
实施例3说明本发明低浓度前处理液
1、制备0.5mol/L、pH7.4磷酸盐缓冲液1000ml;
2、将乙二胺四乙酸二钠1.6g、十二烷基硫酸钠0.2g加入到步骤1得到的磷酸盐缓冲液中,在58℃水浴中加热溶解;
3、将异丁醇3ml加入步骤2制得的溶液中,充分搅拌使之完全溶解;
4、将壳碘55g、甲基异噻唑啉酮0.05ml加入步骤3制得的溶液中,搅拌使之混匀。
实施例4
实施例4说明对照前处理液
基质为0.9%氯化钠溶液,不加入上述化工原料。
试验例1
本试验选用直肠内液血红素蛋白测定作为试验检测项目,以市售标定的《血红素蛋白校准液》为标准,测定不同浓度化工原料配伍(实施例1-4)作为前处理液的检测结果,比较实验误差,优化配制方案。
1、试验方法
(1)试验设备:MYG09-M-J免疫分析仪
(2)检测试剂:苯胺蓝染色液(QA-BAL直肠型),由强安医疗器械有限公司提供。
(3)试验标本:
试验标本1-4:分别取1mg/L《血红素蛋白校准液》0.1ml加入到9.9ml实施例1-4制得的前处理液中混匀,制得10μg/L血红素蛋白液1-4。
(4)操作方法:分别将试验标本1-4标本各200μL、各10份加入至MYG09-M-J免疫分析仪反应杯内,再加入苯胺蓝染色液(QA-BAL直肠型)试剂200μL,37℃温育使试剂与样本充分反应。在反应144秒的时间点上,以光电比色法读取该样本与试剂反应后显色液的吸光度,以此吸光度值通过仪器设定的标准曲线读取相对应的血红素蛋白的含量(μg/L)。
(5)求取4种试验标本各10份之血红素蛋白含量的均值,然后对检测结果的均值进行差异性比较,试验结果如表1:
表1血红素蛋白的含量检测结果
2、统计学处理结果
(1)试验标本2的检测结果与《血红素蛋白校准液》标准数值10.00μg/L比较,P值大于0.05,不具有显著性差异。
(2)试验标本1、3、4的检测结果与《血红素蛋白校准液》标准数值比较,P值均小于0.05,具有显著性差异。
(3)试验标本1、2、3的检测结果与《血红素蛋白校准液》标准数值比较,实验误差均小于25%。
(4)试验标本4未呈现有效检测结果。
3、结果分析:
(1)试验标本1-3的试验结果处于可控范围之内,故实施例1-3提供的配方范围内的任何值均可作为直肠内液样本专用前处理液配伍采用。
(2)实施例2为范围内优化值。
(3)实施例4仅为等渗氯化钠溶液,系空白对照试剂,无直肠内液血红素蛋白测定功能。
4、结论:本发明提供之直肠内液样本专用前处理液配伍合理,能保证试验的精准度。
试验例2
试验例2说明霉菌抑菌试验
试验样本:选用适宜的采样器,在直肠内部位轻轻擦拭,获取直肠内液。采液量为0.5~1ml,一般以采样拭子全部湿润为度。采样后把拭子插入多功能直肠内液样本专用前处理液2ml中轻轻搅动,使拭子上获取的直肠内液与实施例的前处理液混匀,制得样本液。
1、霉菌培养试验方法:
(1)每个试验标本做3个平行实验,以无菌操作技术将直肠内液试验样本0.5ml分别加入平皿内,再分别把试验标本1-4各2ml加到上述平皿内,及时用25ml、45-47℃的孟加拉红琼脂倾注平皿,转动平皿混合均匀。琼脂凝固,正置平板。
(2)28℃培养五天。
(3)根据菌落形态分别计数霉菌,计算每个试验标本平板菌落数的平均值。
2、试验结果:
(1)试验标本1、2、3均未见霉菌生长;
(2)试验标本4霉菌蔓延生长覆盖整个平板,记录为“菌落蔓延”。
3、结论:本发明提供之具有消杀和细胞破碎功能的直肠内液样本专用前处理液能有效地杀灭霉菌。
综上所述,本专利提供的直肠内液样本专用前处理液,能有效地维持直肠内液的稳定性,排除渗液内存在的各种因素的干扰,保证检测的精准度,具有很好的消杀和抑制霉菌和细胞破碎功能,是一种用于检测直肠内液适宜的前处理液。
Claims (6)
1.一种直肠内液样本专用前处理液,其特征在于:每升溶液中包含的组分及含量为:甲基异噻唑啉酮0.05~1.5ml、十二烷基硫酸钠0.2~0.6g、乙二胺四乙酸二钠1.6~3.2g、异丁醇3~7ml、壳聚糖-碘络合物55~80g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
2.根据权利要求1所述的一种直肠内液样本专用前处理液,其特征在于:每升溶液中包含的组分及含量为:甲基异噻唑啉酮0.1ml、十二烷基硫酸钠0.4g、乙二胺四乙酸二钠2g、异丁醇4ml、壳聚糖-碘络合物65g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
3.根据权利要求1所述的一种直肠内液样本专用前处理液,其特征在于:每升溶液中包含的组分及含量为:甲基异噻唑啉酮0.05ml、十二烷基硫酸钠0.2g、乙二胺四乙酸二钠1.6g、异丁醇3ml、壳聚糖-碘络合物55g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
4.根据权利要求1所述的一种直肠内液样本专用前处理液,其特征在于:每升溶液中包含的组分及含量为:甲基异噻唑啉酮1.5ml、十二烷基硫酸钠0.6g、乙二胺四乙酸二钠3.2g、异丁醇7ml、壳聚糖-碘络合物80g和pH值为7.4、浓度为0.5mol磷酸盐缓冲液。
5.根据权利要求1-4任意一项所述的一种直肠内液样本专用前处理液,其特征在于:该前处理液的基质为蒸馏水。
6.一种根据权利要求1-4任意一项所述的直肠内液样本专用前处理液的制备方法,其特征在于:具体步骤如下:
步骤一、磷酸缓冲液的制备:无水磷酸氢二钠加入于蒸馏水中,搅拌至完全溶解,再将无水磷酸二氢钠溶于上液中,定容;
步骤二、将乙二胺四乙酸二钠、十二烷基硫酸钠加入到步骤一得到的磷酸盐缓冲液中,在57-60℃水浴中加热溶解;
步骤三、将异丁醇加入步骤二制得的溶液中,充分搅拌使之完全溶解;
步骤四、将壳碘、甲基异噻唑啉酮加入步骤三制得的溶液中,搅拌使之混匀。
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