CN113956979A - 一种临床痰标本液化处理方法 - Google Patents
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Abstract
本发明公开了一种临床痰标本的液化处理方法。本发明的临床痰液标本液化处理方法采用两步液化法，包括蛋白酶K孵育液化和二硫苏糖醇缓冲液液化。采用本发明的方法可以快速彻底液化临床粘稠痰液，且不损伤痰液中病原微生物与核酸的特点，能够为痰液的微生物培养与核酸提取提供先决条件。
Description
技术领域
本发明涉及临床痰液标本前处理，属于检验医学领域。
背景技术
目前主要应用到临床的痰液标本前处理有：碱裂解法、Saccomanno法和DTT联合法、N-乙酸-L-半胱氨酸-氢氧化钠法。碱裂解法利用碱液使标本中蛋白质发生变性溶解于碱液中，从而达到液化痰液的目的。Saccomanno法和DTT联合法、N-乙酸-L-半胱氨酸-氢氧化钠法通过打开痰液标本中粘蛋白的二硫键增强其水溶性，从而达到液化痰液的目的。
临床痰液标本送检量很大，一些患者的痰液标本中酸性糖蛋白含量很高，而酸性糖蛋白依靠二硫键、氢键等交叉联接在一起形成凝胶网，造成痰液粘稠不易液化。而一份充分液化的痰液，是微生物培养及核酸提取提供先决条件。碱裂解法耗时长，强烈改变痰液标本的PH值，容易导致病原微生物死亡，因此不适合需要做微生物培养的痰液标本，碱裂解往往不充分，容易导致核酸提取效率低下。Saccomanno法和DTT联合法、N-乙酸-L-半胱氨酸-氢氧化钠法能打开粘蛋白中的二硫键，对于一般性状的痰液液化效果好，但痰液中还含有疏水的氢键，这两种方法都无法有效打开氢键，因此对于酸性糖蛋白含量极高的脓性痰液液化时间长，且效果欠佳。
临床需要一种痰液化方法，液化时间短，能够做到标准化，方便处理大批量标本；液化彻底不损伤病原微生物和核酸，液化后的痰液既可以做微生物培养，也可以提取核酸进行分子生物学检测。
发明内容
本发明的一个目的是提供一种临床痰液标本液化处理方法。
本发明所涉及的临床痰液标本液化处理方法是采用两步液化法。所述的两步液化法包括蛋白酶K孵育液化与二硫苏糖醇缓冲液液化。有效的痰液标液化处理方法是微生物培养及核酸提取的关键，只有将痰液标本彻底液化，才能有效复级痰液中的微生物。两步液化法解决了这一技术难点，为临床痰液标本中微生物检测供了可靠的技术保障。
为达到上述目的，本发明的技术方案是这样实现的：
（1）蛋白酶K孵育液化
痰液标本中加入等量20mg/l的蛋白酶K 放入37℃恒温摇床振荡孵育15min;
（2）二硫苏糖醇缓冲液液化
在蛋白酶K孵育液化的基础上再加入等量二硫苏糖醇缓冲液，37℃恒温振荡孵箱孵育20min。
适合浓度的蛋白酶K既可以对痰液中的蛋白质进行液化，又不会破坏微生物的细胞膜，不会影响微生物培养与核酸的提取。而且经蛋白酶K液化后的标本，再进行二硫苏糖醇缓冲液液化更容易液化彻底充分，对粘稠痰液标本效果也很理想。同时两步液化法缩短了液化时间，即使酸性糖蛋白含量极高的浓稠痰液液化时间也能控制在1小时之内。
附图说明
图1—1为碱裂解法痰液（肺炎链球菌）培养结果；
图1—2为Saccomanno法和DTT联合法痰液（肺炎链球菌）培养结果；
图1—3为N-乙酸-L-半胱氨酸-氢氧化钠法痰液（肺炎链球菌）培养结果；
图1—4为蛋白酶K-二硫苏糖醇两步液化法痰液（肺炎链球菌）培养结果。
图2—1为碱裂解法痰液（流感嗜血杆菌）培养结果；
图2—2为Saccomanno法和DTT联合法痰液（流感嗜血杆菌）培养结果；
图2—3为N-乙酸-L-半胱氨酸-氢氧化钠法痰液（流感嗜血杆菌）培养结果；
图2—4为蛋白酶K-二硫苏糖醇两步液化法痰液（流感嗜血杆菌）培养结果。
图3为四种液化方法10份标本核酸平均浓度比较直方图。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以助于理解本发明的内容。
实施例1 试剂及样本的准备
选用下列试剂进行本发明所述的痰液液化处理方法进行微生物培养及核酸提取：
20mg/l的蛋白酶K ，二硫苏糖醇，氯化钾，氯化钠，磷酸氢二钠，磷酸二氢钾，4%氢氧化钠溶液，50%乙醇，0.4%聚乙二醇，0.06%利福平，0.5% N-乙酸-L-半胱氨酸，1.45%枸橼酸钠，2%氢氧化钠溶液。样本选用临床粘稠痰液标本10份，其中5份标本已经确定培养出肺炎链球菌，5份标本已经确定培养出嗜血流感杆菌。将所有样本分成四份，每份分装1ml痰液。
实施例2 临床痰液标本液化处理
（1）方法1:痰标本碱裂解液化法。10份标本各取1ml痰液加入5ml 4%氢氧化钠，37℃恒温振荡60min，高速12000r/min离心5min，去上清加入5ml去离子水振荡洗涤，高速12000r/min，去上清。洗涤过程再重复一次，沉淀备用。
（2）方法2：Saccomanno法和DTT联合痰标本液化法。将Saccomanno液（50%乙醇，0.4%聚乙二醇，0.06%利福平）与DDT液（DDT浓度为0.005%）等体积混合，10份标本各取1ml痰液中加入2ml混合液，振荡60min。
（3）方法3：N-乙酸-L-半胱氨酸-氢氧化钠痰标本液化法。10份标本各取1ml痰液加入2ml N-乙酸-L-半胱氨酸-氢氧化钠液化剂（0.5% N-乙酸-L-半胱氨酸，1.45%枸橼酸钠，2%氢氧化钠）振荡液化60min。
（4）方法4：两步痰标本液化法。10份标本各取1ml痰液标本中加入1ml 20mg/l的蛋白酶K 放入37℃恒温摇床振荡孵育15min。2ml二硫苏糖醇缓冲液，37℃恒温摇床振荡孵育20min。
实施例3 临床痰液标本中病原微生物的培养与核酸提取比较
1、实施例2中的临床痰液四种液化方法处理。
2、临床痰液标本液化后病原微生物定量培养：实施案例2中四种液化方法液化的10份痰液，分别取10µl涂布法接种于巧克力培养基上，5%二氧化碳孵箱35℃孵育48h后观察结果，进行菌落计数。
3、临床痰液标本液化后核酸提取浓度测量：实施案例2中四种液化方法液化的10份痰液分别提取核酸，采用经典的酚-氯仿抽提方法。1ml液化后的痰液加入1ml氯仿异丙醇（氯仿异丙醇比例为24：1）混合液振荡混匀，8000r/min离心10min，取上清加入两倍体积的无水乙醇和10%体积的3mol/L醋酸钠混匀。-20℃冰箱冷冻20min。高速12000r/ml离心15min，去上清沉淀加入1ml 75%酒精振荡混匀，12000r/ml离心10min，去上清，沉淀室温干燥5min后加入100µl Tris-HCL- EDTA缓冲液溶解核酸。利用分光光度测量仪检测提取缓冲液中260nm及280nm光波吸光度的A值，计算核酸浓度。
以上所述仅为本发明的较佳实施例而已，并非用于限定本发明的保护范围。
Claims (3)
1.一种临床痰液标本液化处理方法，包括以下步骤：
（1）蛋白酶K孵育液化；
（2）二硫苏糖醇缓冲液液化。
2.根据权利要求1所述的痰液标本液化处理方法，其特征在于，蛋白酶K孵育液化是在标本中加入等量20mg/l的蛋白酶K 37℃15min。
3.根据权利要求1所述的痰标本液化处理方法，其特征在于，二硫苏糖醇缓冲液液化是在蛋白酶K孵育液化的基础上再加入等量二硫苏糖醇缓冲液，37℃20min，二硫苏糖醇缓冲液配方为100ml无菌蒸馏水中加入二硫苏糖醇0.1g，氯化钾0.02g，氯化钠0.78g，磷酸氢二钠0.112g，磷酸二氢钾0.02g。
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| CN115931503B (zh) * | 2022-12-21 | 2023-12-22 | 江阴天泽医学检验实验室有限公司 | 痰标本简易液化剂和液化装置 |
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