CN115369147A - 一种需氧微生物的培养液及其体外检测试剂盒 - Google Patents

一种需氧微生物的培养液及其体外检测试剂盒 Download PDF

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CN115369147A
CN115369147A CN202211152847.2A CN202211152847A CN115369147A CN 115369147 A CN115369147 A CN 115369147A CN 202211152847 A CN202211152847 A CN 202211152847A CN 115369147 A CN115369147 A CN 115369147A
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吕岚棋
俞海芳
魏永丽
李敏
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ZHEJIANG KUAKE BIOTECHNOLOGY CO Ltd
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Abstract

本发明公开了一种需氧微生物的培养液及其体外检测试剂盒，属于生物化学检测技术领域。所述培养液包括胰蛋白胨、明胶蛋白胨、脑心浸液、L‑精氨酸、牛肉膏、葡萄糖、维生素、硫酸镁、碳酸氢盐、聚茴香脑磺酸、对氨基苯甲酸；所述试剂盒包括培养液、过滤材料、培养瓶瓶体、瓶盖和感受器。该试剂盒中通过培养液各成分之间相互配合，能够有效地促进菌种活性，进而提高需氧微生物的阳性检出率并缩短检出时间，准确性较高；检测需氧微生物的最低检出限能达到50copies/mL，灵敏性较高。

Description

一种需氧微生物的培养液及其体外检测试剂盒
技术领域
本发明属于生物化学检测技术领域，涉及一种需氧微生物的培养液及其体外检测试剂盒。
背景技术
菌血症是指外界的细菌经由体表的入口或是感染的伤口进入血液系统，并在人体血液内繁殖并随血流在全身播散。菌血症多是细菌由局部病灶入血，全身无中毒症状，但血液中可查到细菌，主要发生在炎症的早期阶段。由于菌血症绝大多数继发于各种感染，又缺乏特异的临床表现，故易造成漏诊或误诊。常用血培养的方法来进行以上病症的诊断。血培养是将新鲜离体的血液标本接种于营养培养基上，在一定温度、湿度等条件下，使对营养要求较高的细菌生长繁殖并对其进行鉴别，从而确定病原菌的一种培养法。
中国专利CN 112831539 A公开了一种需氧微生物的体外检测试剂盒，涉及生物化学检测技术领域，该试剂盒包括培养液、吸附树脂、培养瓶瓶体、瓶盖和感受器；其中，培养液包括胰蛋白胨、明胶蛋白胨、脑心浸液、L精氨酸、维生素、枸橼酸钠、磷酸二氢钾、植物甾醇、聚茴香脑磺酸钠和叶酸。该试剂盒能够有效地中和抗生素，为微生物提供了更有利的生长条件，但对微生物的检出效果还有待于进一步提高。
中国专利CN104673662A公开了一种培养瓶及其制备方法及应用，该培养瓶包括瓶体、瓶盖、胶塞、液体培养基、吸附树脂、传感膜，该发明将仪器检测与人工目测合二为一，进行微生物培养时可在瓶外清晰观测微生物生长情况，也可通过仪器实现连续检测，提高了检测灵敏度，缩短了阳性检出时间。但该发明主要实现了荧光和化学传感膜技术相互结合，同时通过吸附树脂的加入降低抗生素的负面影响并通过促生长因子的加入促进维生素的快速繁殖，上述方法均为目前本领域中较为常规的手段，但该发明在培养基成分等方面并未做过多优化，因此有一定的局限性，即难以达到更高阳性检出率以及更短检出时间。
因此，有必要探索一种阳性检出率高、检出时间短的需氧微生物的体外检测试剂盒，从而能够实现需氧微生物的准确、快速检测。
发明内容
针对现有技术存在的问题，本发明的提供了一种需氧微生物的培养液及其体外检测试剂盒。
为实现上述目的，本发明采用的技术方案如下：
首先，提供了一种需氧微生物的培养液，所述培养液包括：胰蛋白胨、明胶蛋白胨、脑心浸液、L-精氨酸、牛肉膏、葡萄糖、维生素、硫酸镁、碳酸氢盐、聚茴香脑磺酸、对氨基苯甲酸。
进一步地，所述培养液包括：1-10g/L胰蛋白胨、1-10g/L明胶蛋白胨、1-10g/L脑心浸液、1-10g/L L-精氨酸、1-10g/L牛肉膏、1-10g/L葡萄糖、0.1-0.15g/L维生素、0.2-0.5g/L硫酸镁、0.1-0.3g/L碳酸氢盐、0.04-0.06g/L聚茴香脑磺酸、0.008-0.02g/L对氨基苯甲酸。
进一步地，所述硫酸镁、碳酸氢盐的重量比为0.2-0.45：0.1-0.25。
进一步地，所述聚茴香脑磺酸、对氨基苯甲酸的重量比为0.04-0.055：0.01-0.02。
进一步地，所述碳酸氢盐包括碳酸氢钠、碳酸氢钾、碳酸氢钙、碳酸氢铵的一种或几种，优选为碳酸氢钠。
进一步地，所述维生素包括维生素A、维生素B、维生素D、维生素E的一种或几种，优选为维生素A、维生素B。
进一步地，所述培养液使用双蒸水进行配制。
其次，提供了一种包括所述培养液的试剂盒，所述试剂盒还包括过滤材料、培养瓶瓶体、瓶盖和感受器。
进一步地，所述过滤材料为过滤膜，所述过滤膜为聚醚砜、聚砜、混合纤维素酯、聚碳酸酯的任意两种，优选为聚醚砜、聚砜、聚碳酸酯的任意两种。
进一步地，所述感受器位于培养瓶瓶体底部，含有荧光染料。
最后，提供了一种所述的培养液或所述的试剂盒在非诊断目的体外检测需氧微生物中的应用。
进一步地，所述需氧微生物包括肺炎链球菌、铜绿假单胞菌、粪产碱杆菌、化脓链球菌、白色念珠菌、流感嗜血杆菌、大肠杆菌、金黄色葡萄球菌和脑膜炎奈瑟氏菌。
相比于现有技术，本发明具有以下有益效果：
(1)本发明中产品的液体培养基中加入多种营养成分，可以供多种微生物的生长培养，硫酸镁、碳酸氢盐之间、聚茴香脑磺酸、对氨基苯甲酸之间通过相互配合，能够有效地促进菌种活性，非但不会抑制菌群生长，还能够在一定程度上提高培养阳性率；
(2)本发明所提供的试剂盒能够有效地缩短肺炎链球菌、铜绿假单胞菌、粪产碱杆菌和化脓链球菌的检出时间，提高阳性检出率，准确性较高；
(3)本发明所提供的试剂盒检测肺炎链球菌、铜绿假单胞菌、粪产碱杆菌和化脓链球菌4种需氧菌的最低检出限均能达到50copies/mL，灵敏性较高。
具体实施方式
值得说明的是，本发明中使用的原料均为普通市售产品，对其来源不做具体限定。
实施例1
一种需氧微生物的体外检测试剂盒，包括培养液、过滤材料、培养瓶瓶体、瓶盖和感受器；
其中，培养液包括1g/L胰蛋白胨、1g/L明胶蛋白胨、1g/L脑心浸液、1g/L L-精氨酸、1g/L牛肉膏、1g/L葡萄糖、0.05g/L维生素A、0.05g/L维生素B、0.2g/L硫酸镁、0.1g/L碳酸氢钠、0.04g/L聚茴香脑磺酸、0.008g/L对氨基苯甲酸。上述培养液使用双蒸水进行配制，感受器位于培养瓶瓶体底部，含有荧光染料，过滤材料为体积比为1.5：1的聚醚砜和聚碳酸酯。
实施例2
一种需氧微生物的体外检测试剂盒，包括培养液、过滤材料、培养瓶瓶体、瓶盖和感受器；
其中，培养液包括10g/L胰蛋白胨、10g/L明胶蛋白胨、10g/L脑心浸液、10g/L L-精氨酸、10g/L牛肉膏、10g/L葡萄糖、0.05g/L维生素A、0.1g/L维生素B、0.5g/L硫酸镁、0.3g/L碳酸氢钠、0.06g/L聚茴香脑磺酸、0.02g/L对氨基苯甲酸。上述培养液使用双蒸水进行配制，感受器位于培养瓶瓶体底部，含有荧光染料，过滤材料为体积比为1.5：1的聚砜和聚碳酸酯。
实施例3
一种需氧微生物的体外检测试剂盒，包括培养液、过滤材料、培养瓶瓶体、瓶盖和感受器；
其中，培养液包括5g/L胰蛋白胨、5g/L明胶蛋白胨、5g/L脑心浸液、5g/L L-精氨酸、5g/L牛肉膏、5g/L葡萄糖、0.05g/L维生素A、0.05g/L维生素B、0.3g/L硫酸镁、0.2g/L碳酸氢钠、0.05g/L聚茴香脑磺酸、0.015g/L对氨基苯甲酸。上述培养液使用双蒸水进行配制，感受器位于培养瓶瓶体底部，含有荧光染料，过滤材料为体积比为1.5：1的聚醚砜和聚碳酸酯。
对比例1
与实施例1的区别仅在于，培养液包括0.8g/L胰蛋白胨、11g/L明胶蛋白胨、0.8g/L脑心浸液、11g/L L-精氨酸、0.8g/L牛肉膏、11g/L葡萄糖、0.04g/L维生素A、0.04g/L维生素B、0.6g/L硫酸镁、0.08g/L碳酸氢钠、0.08g/L聚茴香脑磺酸、0.006g/L对氨基苯甲酸。
对比例2
与实施例1的区别仅在于，不加入硫酸镁，碳酸氢钠在培养液中的浓度为0.3g/L。
对比例3
与实施例1的区别仅在于，不加入碳酸氢钠，硫酸镁在培养液中的浓度为0.3g/L。
对比例4
与实施例1的区别仅在于，不加入聚茴香脑磺酸，对氨基苯甲酸在培养液中的浓度为0.048g/L。
对比例5
与实施例1的区别仅在于，不加入对氨基苯甲酸，聚茴香脑磺酸在培养液中的浓度为0.048g/L。
试验1：本发明中试剂盒的准确性
选前四种需氧菌(肺炎链球菌、铜绿假单胞菌、粪产碱杆菌、化脓链球菌)，采用不同实例中的试剂盒进行检测，每种各测试20组，统计阳性检出率(菌悬液浓度为100cfu/mL)，得到表1和表2。
表1
Figure BDA0003857069920000051
表2
Figure BDA0003857069920000052
本发明的试剂盒能够有效地缩短肺炎链球菌、铜绿假单胞菌、粪产碱杆菌和化脓链球菌的检出时间，提高阳性检出率，其中，实施例1-3中试剂盒对于各菌种的检出时间介于7.2-10.2h，阳性检出率可达100％。相比较而言，各组对比例由于培养液中原料组成成分的选择以及配比情况未达到合适状态，最终检出时间较长，阳性检出率也未达到期许值。
试验2：本发明中试剂盒的灵敏性
选前四种需氧菌(肺炎链球菌、铜绿假单胞菌、粪产碱杆菌、化脓链球菌)，分别配制成103copies/mL、102copies/mL、50copies/mL、10copies/mL的检测样本，采用本发明不同实例中的试剂盒，检测四种需氧菌的最低检出限，结果如表3所示。
表3最低检出限试验结果
Figure BDA0003857069920000061
注：+表示对103(或102或50或10)copies/mL的检测样本重复检测25次，25次检测均为阳性；-表示25次检测至少一次为阴性。
由表3可知，实施例1-3的试剂盒检测4种需氧菌的最低检出限均能达到50copies/mL。
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。

Claims (10)

1.一种需氧微生物的培养液，其特征在于，所述培养液包括：胰蛋白胨、明胶蛋白胨、脑心浸液、L-精氨酸、牛肉膏、葡萄糖、维生素、硫酸镁、碳酸氢盐、聚茴香脑磺酸、对氨基苯甲酸。
2.根据权利要求1所述的培养液，其特征在于，所述培养液包括：1-10g/L胰蛋白胨、1-10g/L明胶蛋白胨、1-10g/L脑心浸液、1-10g/L L-精氨酸、1-10g/L牛肉膏、1-10g/L葡萄糖、0.1-0.15g/L维生素、0.2-0.5g/L硫酸镁、0.1-0.3g/L碳酸氢盐、0.04-0.06g/L聚茴香脑磺酸、0.008-0.02g/L对氨基苯甲酸。
3.根据权利要求1所述的培养液，其特征在于，所述硫酸镁、碳酸氢盐的重量比为0.2-0.45：0.1-0.25。
4.根据权利要求1所述的培养液，其特征在于，所述聚茴香脑磺酸、对氨基苯甲酸的重量比为0.04-0.055：0.01-0.02。
5.根据权利要求1所述的培养液，其特征在于，所述碳酸氢盐包括碳酸氢钠、碳酸氢钾、碳酸氢钙、碳酸氢铵的一种或几种。
6.一种包括权利要求1-5任一项所述培养液的试剂盒，其特征在于，所述试剂盒还包括过滤材料、培养瓶瓶体、瓶盖和感受器。
7.根据权利要求6所述的试剂盒，其特征在于，所述过滤材料为过滤膜，所述过滤膜为聚醚砜、聚砜、混合纤维素酯、聚碳酸酯的任意两种。
8.根据权利要求6所述的试剂盒，其特征在于，所述感受器位于培养瓶瓶体底部，含有荧光染料。
9.如权利要求1-5任一项所述的培养液或权利要求6-8任一项所述的试剂盒在非诊断目的体外检测需氧微生物中的应用。
10.根据权利要求9所述的应用，其特征在于，所述需氧微生物包括肺炎链球菌、铜绿假单胞菌、粪产碱杆菌、化脓链球菌、白色念珠菌、流感嗜血杆菌、大肠杆菌、金黄色葡萄球菌和脑膜炎奈瑟氏菌。
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