WO2022068751A1 - 一种痰液样本的消化方法 - Google Patents

一种痰液样本的消化方法 Download PDF

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WO2022068751A1
WO2022068751A1 PCT/CN2021/120824 CN2021120824W WO2022068751A1 WO 2022068751 A1 WO2022068751 A1 WO 2022068751A1 CN 2021120824 W CN2021120824 W CN 2021120824W WO 2022068751 A1 WO2022068751 A1 WO 2022068751A1
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sputum
digesting
peptone
sputum sample
surfactant
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PCT/CN2021/120824
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English (en)
French (fr)
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王则宇
杨咏康
高静
张晨阳
丁朋举
李晓霞
孙武举
付光宇
吴学炜
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郑州安图生物工程股份有限公司
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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
    • 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
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising

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  • the invention relates to a digestion method for sputum samples, belonging to the technical field of sputum digestion.
  • Sputum samples, urine, secretions, feces, etc. are common clinical samples. Among them, sputum samples are currently the largest samples in China, accounting for about 70% of the total samples. Sputum samples are semi-solid and heterogeneous samples, and pathogenic bacteria are often encapsulated in mucus. Random sampling and direct testing can easily lead to missed or false detections. Therefore, sputum samples need to be bacteriologically cultured before testing. Homogenization of sputum samples (commonly known as sputum digestion) is an important step in sputum bacteriological culture.
  • Homogenization of sputum samples is the incubation and digestion of sputum samples after adding sputum sample digestive juices to the sputum samples.
  • the incubation and digestion time is usually long (up to 1 hour), which is not conducive to rapid inoculation.
  • the shaking of the sample added to the sputum digestive juice can achieve rapid digestion, but the shaking and digestion can easily lead to bacterial lysis and death, which in turn affects the accuracy of the test results.
  • the purpose of the present invention is to provide a sputum sample digestion method, which can solve the problem that microorganisms easily die in large numbers when the sputum sample is added to the digestive juice for shock digestion in the existing digestion method.
  • a method for digesting a sputum sample comprising the steps of: shaking and digesting a mixture of a sputum sample, a sputum digestive juice and a shock protection agent; the shock protection agent is selected from defoamer, surfactant, protein, peptone, Any one or combination of polyhydroxy organics.
  • the sputum sample digestion method of the present invention by shaking and digesting the mixture of the sputum sample digestive juice, the sputum sample and the shock protection agent, can not only shorten the digestion time, but also ensure the digestion effect of the sputum sample. During the shaking process, it will not be cracked or die, and the accuracy of the inoculation results will be improved.
  • the shock protection agent used in the sputum sample digestion method of the present invention can be one of defoaming agent, surfactant, protein, peptone, and polyhydroxy organic, or can be any combination, such as defoaming agent + surfactant , protein + surfactant, peptone + surfactant, polyhydroxy organic matter + surfactant, defoamer + protein + surfactant, etc.
  • the sputum digestive liquid used can be any existing sputum digestive liquid, including commercially available products, such as pancreatin sputum digestive liquid, ⁇ -chymotrypsin sputum digestive liquid, N-acetyl-L-cysteine sputum digestive liquid, Sputasol Sputum digestive juice, sputum digestive juice containing DTT, or rapid digestion liquid of sputum samples, etc., are used according to the instructions.
  • the shaking digestion time is 5-15 min.
  • the time of shaking digestion can be 7min, 9min, 10min, 12min or 14min.
  • the mixture is obtained by mixing the oscillating protective agent and the sputum digestive juice to prepare a compound sputum digestive juice, and then mixing the compound sputum digestive juice and a sputum sample.
  • the volume ratio of the sputum sample to the composite sputum digestive juice is preferably 1:1 to 1:2.
  • the mixture can also be obtained by adding the sputum digestive juice and the shaking protective agent to the sputum sample respectively for mixing.
  • the defoamer is a silicon-free defoamer.
  • the silicon-free defoamer is preferably a fermented food grade silicon-free defoamer.
  • the silicon-free defoamer can be one or both of the silicon-free defoamer F1152 and the silicon-free defoamer DF-103.
  • the surfactant is a nonionic surfactant.
  • the nonionic surfactant is selected from any one or a combination of polyoxyethylene type nonionic surfactants and polyol type nonionic surfactants.
  • the polyoxyethylene type nonionic surfactant is polyethylene glycol. The average molecular weight of the polyethylene glycol is 2000-6000.
  • the polyoxyethylene type nonionic surfactant is any one or a combination of polyethylene glycol 2000 and polyethylene glycol 6000.
  • the polyol type nonionic surfactant is a Tween type nonionic surfactant.
  • the polyol type nonionic surfactant is selected from one or both of Tween-20 and Tween-80.
  • the polyhydroxy organic substance is a carbohydrate substance.
  • the saccharide substances can be one or any combination of disaccharides and polysaccharides.
  • the carbohydrate substance is selected from any one or a combination of dextrin, trehalose and sucrose.
  • the protein is any one or a combination of bovine serum albumin and casein.
  • the peptone is any one or a combination of animal-derived peptone and plant-derived peptone.
  • the peptone is animal-derived peptone.
  • the mass of the shock protective agent used for each 1 L of sputum digestive liquid is 0.01-41 g.
  • the mass of the shock protectant used for each 1L of sputum digestive juice can be 0.05g, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 31g, 32g, 33g, 34g, 35g, 36g, 37g, 38g, 39g or 40g.
  • the shock protection agent includes an antifoaming agent.
  • the mass of the antifoaming agent used for each 1 L of sputum digestive liquid is 0.01-1 g.
  • the mass of the antifoaming agent used for each 1L of sputum digestive juice may be 0.05g, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g or 0.9g.
  • the shock protective agent can be a combination of defoaming agent or at least one of surfactants, proteins, peptones, and polyhydroxy organics and defoaming agents, such as defoaming agent + surface Active agent, defoamer + protein, defoamer + peptone, defoamer + polyhydroxy organic matter, another example of defoamer + surfactant + protein, defoamer + surfactant + peptone, defoamer + surface Active agent + polyhydroxy organic matter, defoamer + protein + peptone, defoamer + protein + polyhydroxy organic matter, defoamer + peptone + polyhydroxy organic matter, and then defoamer + surfactant + protein + peptone, antifoam Foaming agent + surfactant + protein + polyhydroxy organic matter, defoamer + protein + peptone + polyhydroxy organic matter, defoaming agent + surfactant + protein + peptone, defoaming agent + surfactant +
  • the shock protection agent includes a surfactant.
  • the mass of the surfactant corresponding to each 1 L of sputum digestive juice is 0.1-10 g.
  • the mass of the surfactant used for each 1 L of sputum digestive juice may be 0.5 g, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g or 9 g.
  • the shock protection agent can be a combination of at least one of a surfactant or a defoamer, a protein, a peptone, and a polyhydroxy organic compound and a surfactant, such as surfactant + protein, surface Active agent + peptone, surfactant + polyhydroxy organic matter, such as surfactant + protein + peptone, surfactant + protein + polyhydroxy organic matter, surfactant + peptone + polyhydroxy organic matter, surfactant + protein + peptone + Polyhydroxy organics.
  • a surfactant or a defoamer such as surfactant + protein, surface Active agent + peptone, surfactant + polyhydroxy organic matter, such as surfactant + protein + peptone, surfactant + protein + polyhydroxy organic matter, surfactant + peptone + polyhydroxy organic matter, surfactant + protein + peptone + Polyhydroxy organics.
  • a surfactant or a defoamer such as surfactant + protein, surface Active agent + peptone, surfactant +
  • the shock protectant includes protein.
  • the mass of the protein used for each 1 L of sputum digestive juice is 1-10 g.
  • the mass of the protein used for each 1 L of sputum digestion solution may be 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g or 9 g.
  • the shock protective agent can be a combination of protein or at least one of defoaming agent, surfactant, peptone, polyhydroxy organic matter and protein, such as protein + peptone, protein + polyhydroxy organic matter, protein. + peptone + polyhydroxy organics.
  • the shock protection agent includes peptone.
  • the mass of peptone used for each 1 L of sputum digestion solution is 1-10 g.
  • the mass of peptone used for each 1 L of sputum digestion solution may be 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g or 9 g.
  • the shock protective agent may be a combination of peptone or at least one of antifoaming agent, surfactant, protein, and polyhydroxy organic matter with peptone, such as peptone + polyhydroxy organic matter.
  • the shock protection agent includes a polyhydroxy organic compound.
  • the mass of the polyhydroxy organic matter correspondingly used per 1 L of sputum digestive juice is 1-10 g.
  • the mass of the polyhydroxy organic matter used per 1 L of sputum digestive juice may be 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g or 9 g.
  • the shock protective agent may be a polyhydroxy organic compound or a combination of at least one of a defoaming agent, a surfactant, a protein, and a peptone and a polyhydroxy organic compound.
  • Fig. 1 is the contrast diagram before and after the shock of sputum sample in Experimental Example 2;
  • Fig. 2 is the contrast diagram before and after the shock of sputum sample in Experimental Example 3;
  • Fig. 3 is the contrast diagram before and after the shock of sputum sample in Experimental Example 4;
  • Figure 4 is a comparison diagram of the sputum sample before and after shaking in Experimental Example 5.
  • the animal-derived peptones used in the examples and experimental examples were bacteriological peptones purchased from Yanshi Baijia Industry and Trade Co., Ltd.
  • Antu biological sputum digestive liquid is the commercially available sputum sample digestive liquid produced by Zhengzhou Antu Bioengineering Co., Ltd. (No. 20190184 of Yuzheng Machinery, the main component is DTT), and the OXOID sputum digestive liquid adopted is OXOID The company's sputum digestive juice (Sputasol) products.
  • Silicon-free defoamer F1152 and silicon-free defoamer DF-103 used in Examples and Experimental Examples were purchased from Shenzhen Dayang New Materials Co., Ltd.
  • the method for digesting a sputum sample in this embodiment includes the following steps: taking 1 mL of the sputum sample, adding 1 mL of a composite sputum digestive liquid containing a shock protection agent, and then shaking the sample at room temperature for 10 minutes.
  • the composite phlegm digestive liquid used in this example is obtained by adding shock protectant to the sputum digestive liquid and mixing; the phlegm digestive liquid used is Antu biological phlegm digestive liquid, and the shock protector is silicon-free antifoaming agent F1152, and every 1 L of Antu
  • the mass of the vibration protective agent added to the biological phlegm digestive juice is 0.1 g.
  • the method for digesting a sputum sample in this embodiment includes the following steps: taking 1 mL of the sputum sample, adding 1 mL of a composite sputum digestive liquid containing a shock protection agent, and then shaking the sample at room temperature for 10 minutes.
  • the composite phlegm digestive liquid used in this example is obtained by adding a shock protective agent to Antu biological phlegm digestive liquid and mixing; the shock protective agent used is Tween-80, and the shock protective agent added in every 1 L of Antu biological phlegm digestive liquid
  • the mass is 1g.
  • the shock protection agents used in this example are silicon-free antifoaming agent F1152 and Tween-80.
  • the mass of the silicon-free defoamer F1152 is 0.1g, and the mass of Tween-80 added to each 1L of Antu Biological Sputum Digestive Liquid is 1g.
  • the sputum digestion liquid used in this embodiment is OXOID sputum digestion liquid
  • the shock protection agent is sucrose
  • the shaking added in each 1L of OXOID sputum digestion liquid The mass of the protective agent is 1 g.
  • the only difference between the digestion method of the sputum sample in this example and Example 1 is that the shock protective agent used in this example is animal-derived peptone, and the mass of the shock protective agent added to each 1 L of Antu Biological Sputum Digestive Liquid is 5g .
  • the digestion method of sputum samples of Examples 6-52 includes the following steps: taking 1 mL of sputum samples, adding 1 mL of compound sputum digestive juice containing shock protection agent, and then shaking for 10 min in a room temperature environment.
  • the adopted compound phlegm digestive juice is obtained by adding shock protection agent to the sputum digestive juice and mixing.
  • the consumption of the phlegm digestive juice, shock protection agent and shock protection agent adopted in concrete each embodiment is shown in Table 1.
  • the compound phlegm digestive liquid is obtained by adding the shock protective agent to Antu biological phlegm digestive liquid and mixing; the quality of the shock protective agent added to each 1L of Antu biological phlegm digestive liquid is 1 g; the shock protective agent used is Tween- 80.
  • the morphology of each type of sample before and after shaking is shown in Figure 1.
  • the first row in the first column is a schematic diagram of the first bacterial suspension obtained for the first time without shaking. Schematic diagram of the first bacterial suspension obtained in two repetitions after adding physiological saline and shaking.
  • the first row in the second column is a schematic diagram of the second bacterial suspension obtained for the first time after adding Antu biological sputum digestion solution without shaking, and the second to third rows in the second column are the second batch obtained after two repetitions Schematic diagram of the bacterial suspension added to Antu Biological Sputum Digestive Solution after shaking.
  • the first row in the third column is a schematic diagram of the third bacterial suspension obtained for the first time by adding the compound sputum digestive solution without shaking
  • the second to third rows in the third column are the third bacterial suspension obtained after two repetitions.
  • the shock itself caused the death of the bacterial colonies
  • the bacterial colonies of the Antu biological phlegm digestive juice with the shock protective agent added were significantly more than those without the shock protective agent.
  • the compound phlegm digestive liquid is obtained by adding the shock protective agent to Antu biological phlegm digestive liquid and mixing, and the mass of the shock protective agent added in every 1 L of Antu biological phlegm digestive liquid is 1 g; the shock protective agent used is Tween- 80.
  • the morphology of each type of sample before and after shaking is shown in Figure 2.
  • the first row in the first column is a schematic diagram of the first bacterial suspension obtained for the first time adding the compound sputum digestive solution without shaking, and the second to third rows in the first column are shown in Figure 2.
  • the first row in the second column is a schematic diagram of the second bacterial suspension obtained for the first time by adding physiological saline without shaking, and the second to third rows in the second column are the second bacterial suspension obtained after two repetitions. Schematic diagram after adding normal saline and shaking.
  • the first row in the third column is a schematic diagram of the third bacterial suspension obtained for the first time after adding Antu biological sputum digestion solution without shaking, and the second to third rows in the third column are the third batch obtained after two repetitions.
  • the compound phlegm digestive solution is obtained by adding shock protection agent to OXOID phlegm digestive solution and mixing; the shock protection agent used is Tween-80 + silicon-free antifoaming agent F1152, and the silicon-free anti-foaming agent added to each 1L of OXOID digestive solution
  • the mass of foaming agent F1152 is 0.1 g, and the mass of added Tween-80 is 1 g.
  • FIG. 3 The morphology of each type of samples before and after shaking is shown in Figure 3, where row 1 in column 1 is a schematic diagram of the first bacterial suspension obtained for the first time adding compound sputum digestive solution without shaking, and rows 2 to 3 in column 1 Schematic diagram of the first bacterial suspension obtained when the behavior was repeated twice after adding the compound sputum digestive juice and shaking.
  • the first row in the second column is a schematic diagram of the second bacterial suspension obtained for the first time by adding physiological saline without shaking, and the second to third rows in the second column are the second bacterial suspension obtained after two repetitions. Schematic diagram after adding normal saline and shaking.
  • the first row in the third column is a schematic diagram of the third bacterial suspension obtained for the first time after adding the OXOID sputum digestive solution without shaking
  • the second to third rows in the third column are the third bacterial suspension obtained after two repetitions.
  • Schematic diagram of adding OXOID sputum digestive juice after shaking It can be seen from Figure 3 that the shock itself caused the colony to die, and the OXOID sputum digestive juice with shock protection agent was shaken and the number of colonies was significantly higher than that without shock protection agent.
  • the compound phlegm digestive solution is obtained by adding shock protection agent to OXOID phlegm digestive solution and mixing; the shock protection agent used is Tween-80 + silicon-free antifoaming agent F1152, and the silicon-free anti-foaming agent added to each 1L of OXOID digestive solution
  • the mass of foaming agent F1152 is 0.1 g, and the mass of added Tween-80 is 2 g.
  • FIG. 4 The morphology of each type of sample before and after shaking is shown in Figure 4, in which the first row in the first column is a schematic diagram of the first bacterial suspension obtained for the first time adding the compound sputum digestive solution without shaking, and the second to third in the first column. Schematic diagram of the first bacterial suspension obtained when the behavior was repeated twice after adding the compound sputum digestive juice and shaking.
  • the first row in the second column is a schematic diagram of the second bacterial suspension obtained for the first time by adding physiological saline without shaking, and the second to third rows in the second column are the second bacterial suspension obtained after two repetitions. Schematic diagram after adding normal saline and shaking.
  • the first row in the third column is a schematic diagram of the third bacterial suspension obtained for the first time after adding the OXOID sputum digestive solution without shaking
  • the second to third rows in the third column are the third bacterial suspension obtained after two repetitions.
  • Schematic diagram of adding OXOID sputum digestive juice after shaking It can be seen from Figure 4 that the shock itself caused the colony to die, and the OXOID sputum digestive juice with shock protective agent was shaken and the colony was significantly more than the OXOID sputum digestive liquid without shock protective agent.

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Abstract

一种痰液样本的消化方法，包括以下步骤：将痰液样本、痰消化液和震荡保护剂的混合物进行震荡消化；震荡保护剂选自消泡剂、表面活性剂、蛋白质、蛋白胨、多羟基有机物中的任意一种或组合。该方法不仅能够缩短消化时间，还可以在保证痰液样本消化效果的基础上，确保微生物在震荡过程中不被裂解、死亡，提高接种结果的准确性。

Description

一种痰液样本的消化方法 技术领域
本发明涉及一种痰液样本的消化方法，属于痰消化技术领域。
背景技术
痰液样本、尿液、分泌物、粪便等是临床上常见样本。其中，痰液样本是目前国内样本量最大的样本，占样本总量的70％左右。痰液样本是半固体状的非均一性样本，致病菌常常被包裹在粘液中，随机取样直接检验易造成漏检或误检，因此需要对痰液样本进行细菌学培养后再进行检验。痰液样本均质化(俗称痰消化)是痰液细菌学培养的重要步骤。
痰液样本均质化是在痰液样本中加入痰液样本消化液后进行温育消化。为保证消化效果，温育消化时间通常较久(长达1小时)，不利于快速接种。而对加入痰消化液的样本进行震荡可以实现快速消化，但震荡消化易导致细菌裂解死亡，进而影响检测结果的准确性。
发明内容
本发明的目的是提供一种痰液样本的消化方法，能够解决现有消化方法将痰液样本加入消化液进行震荡消化时微生物易大量死亡的问题。
为了实现以上目的，本发明所采用的技术方案是：
一种痰液样本的消化方法，包括以下步骤：将痰液样本、痰消化液和震荡保护剂的混合物进行震荡消化；所述震荡保护剂选自消泡剂、表面活性剂、蛋白质、蛋白胨、多羟基有机物中的任意一种或组合。
本发明的痰液样本的消化方法，将痰样本消化液、痰液样本和震荡保护剂的混合物进行震荡消化，不仅能够缩短消化时间，还可以在保证痰样本消化效果的基础上，确保微生物在震荡过程中不被裂解、死亡，提高接种结果的准确性。
本发明的痰液样本的消化方法采用的震荡保护剂可以为消泡剂、表面活性剂、蛋白质、蛋白胨、多羟基有机物中的一种，也可以是任意组合，如消泡剂+表面活性剂、蛋白质+表面活性剂、蛋白胨+表面活性剂、多羟基有机物+表面活性剂、消泡剂+蛋白质+表面活性剂等。采用的痰消化液可以为任意的现有痰消化液，包括市售产品，如胰酶痰消化液、α-糜蛋白酶痰消化液、N-乙酰-L-半胱氨酸痰消化液、Sputasol痰消化液、含DTT的痰消化液或痰样本快速消化液等，根据说明书使用。
优选的，所述震荡消化的时间为5～15min。例如震荡消化的时间可以7min、9min、10min、12min或14min。
进一步的，所述混合物是将振荡保护剂和痰消化液进行混合制成复合痰消化液，然后再将复合痰消化液和痰液样本进行混合得到。痰液样本与复合痰消化液的体积比优选为1:1～1:2。此外，所述混合物也可以是分别将痰消化液和振荡保护剂加入痰液样本中进行混合得到。
优选的，所述消泡剂为无硅消泡剂。所述无硅消泡剂优选为发酵食品级无硅消泡剂。例如无硅消泡剂可以为无硅消泡剂F1152、无硅消泡剂DF-103中的一种或两种。
优选的，所述表面活性剂为非离子表面活性剂。进一步优选的，所述非离子表面活性剂选自聚氧乙烯型非离子表面活性剂、多元醇型非离子表面活性剂中的任意一种或组合。例如所述聚氧乙烯型非离子表面活性剂为聚乙二醇。所述聚乙二醇的平均分子量为2000～6000。优选的，所述聚氧乙烯型非离子表面活性剂为聚乙二醇2000、聚乙二醇6000中的任意一种或组合。例如所述多元醇型非离子表面活性剂为吐温型非离子表面活性剂。优选的，所述多元醇型非离子表面活性剂选自吐温-20、吐温-80中的一种或两种。
优选的，所述多羟基有机物为糖类物质。糖类物质可以为双糖、多糖中的一种或任意组合。优选的，所述糖类物质选自糊精、海藻糖、蔗糖中的任意一种或组合。
优选的，所述蛋白质为牛血清白蛋白、酪蛋白中的任意一种或组合。
所述蛋白胨为动物源蛋白胨、植物源蛋白胨中的任意一种或组合。优选的，所述蛋白胨为动物源蛋白胨。
优选的，每1L痰消化液对应使用的震荡保护剂的质量为0.01～41g。例如每1L痰消化液对应使用的震荡保护剂的质量可以为0.05g、0.1g、0.2g、0.3g、0.4g、0.5g、0.6g、0.7g、0.8g、0.9g、1g、2g、3g、4g、5g、6g、7g、8g、9g、10g、11g、12g、13g、14g、15g、16g、17g、18g、19g、20g、21g、22g、23g、24g、25g、26g、27g、28g、29g、30g、31g、32g、33g、34g、35g、36g、37g、38g、39g或40g。
优选的，所述震荡保护剂包括消泡剂。进一步优选的，每1L痰消化液对应使用的消泡剂的质量为0.01～1g。例如每1L痰消化液对应使用的消泡剂的质量可以为0.05g、0.1g、0.2g、0.3g、0.4g、0.5g、0.6g、0.7g、0.8g或0.9g。所述震荡保护剂包括消泡剂时，震荡保护剂可以为消泡剂或表面活性剂、蛋白质、蛋白胨、多羟基有机物中的至少要一种与消泡剂的组合，如消泡剂+表面活性剂、消泡剂+蛋白质、消泡剂+蛋白胨、消泡剂+多羟基有机物，又如消泡剂+表面活性剂+蛋白质、消泡剂+表面活性剂+蛋白胨、消泡剂+表面活性剂+多羟基有机物、消泡剂+蛋白质+蛋白胨、消泡剂+蛋白质+多羟基有机物、消泡剂+蛋白胨+多羟基有机物，再如消泡剂+表面活性剂+蛋白质+蛋白胨、消泡剂+表面活性剂+蛋白质+多羟基有机物、消泡剂 +蛋白质+蛋白胨+多羟基有机物、消泡剂+表面活性剂+蛋白质+蛋白胨+多羟基有机物。
优选的，所述震荡保护剂包括表面活性剂。进一步优选的，每1L痰消化液对应使用的表面活性剂的质量为0.1～10g。例如每1L痰消化液对应使用的表面活性剂的质量可以为0.5g、1g、2g、3g、4g、5g、6g、7g、8g或9g。所述震荡保护剂包括表面活性剂时，震荡保护剂可以为表面活性剂或消泡剂、蛋白质、蛋白胨、多羟基有机物中至少一种与表面活性剂的组合，如表面活性剂+蛋白质、表面活性剂+蛋白胨、表面活性剂+多羟基有机物，又如表面活性剂+蛋白质+蛋白胨、表面活性剂+蛋白质+多羟基有机物、表面活性剂+蛋白胨+多羟基有机物、表面活性剂+蛋白质+蛋白胨+多羟基有机物。
优选的，所述震荡保护剂包括蛋白质。进一步优选的，每1L痰消化液对应使用的蛋白质的质量为1～10g。例如每1L痰消化液对应使用的蛋白质的质量可以为1g、2g、3g、4g、5g、6g、7g、8g或9g。所述震荡保护剂包括蛋白质时，震荡保护剂可以为蛋白质或消泡剂、表面活性剂、蛋白胨、多羟基有机物中至少一种与蛋白质的组合，例如蛋白质+蛋白胨、蛋白质+多羟基有机物、蛋白质+蛋白胨+多羟基有机物。
优选的，所述震荡保护剂包括蛋白胨。进一步优选的，每1L痰消化液对应使用的蛋白胨的质量为1～10g。例如每1L痰消化液对应使用的蛋白胨的质量可以1g、2g、3g、4g、5g、6g、7g、8g或9g。所述震荡保护剂包括蛋白胨时，震荡保护剂可以为蛋白胨或消泡剂、表面活性剂、蛋白质、多羟基有机物中至少一种与蛋白胨的组合，例如蛋白胨+多羟基有机物。
优选的，所述震荡保护剂包括多羟基有机物。进一步优选的，每1L痰消化液对应使用的多羟基有机物的质量为1～10g。例如每1L痰消化液对应使用的多羟基有机物的质量可以为1g、2g、3g、4g、5g、6g、7g、8g或9g。所述震荡保护剂包括多羟基有机物时，振荡保护剂可以为多羟基有机物或消泡剂、表面活性剂、蛋白质、蛋白胨中至少一种与多羟基有机物的组合。
附图说明
图1为实验例2中痰液样本震荡前后的对比图；
图2为实验例3中痰液样本震荡前后的对比图；
图3为实验例4中痰液样本震荡前后的对比图；
图4为实验例5中痰液样本震荡前后的对比图。
具体实施方式
以下结合具体实施方式本发明的技术方案作进一步的说明。
实施例以及实验例中采用的动物源蛋白胨为购自偃师百家工贸有限公司的细菌学蛋 白胨。
实施例以及实验例中安图生物痰消化液为郑州安图生物工程股份有限公司生产的市售痰样本消化液(豫郑械备20190184号，主要成分为DTT)，采用的OXOID痰消化液为OXOID公司生产的痰消化液(Sputasol)产品。
实施例以及实验例中采用的无硅消泡剂F1152、无硅消泡剂DF-103均购自深圳大洋新材料有限公司。
实施例1
本实施例的痰液样本的消化方法，包括以下步骤：取痰液样本1mL，加1mL含震荡保护剂的复合痰消化液，然后在室温的环境中震荡10min。
本实施例采用的复合痰消化液是将震荡保护剂加入痰消化液中混合得到；采用的痰消化液为安图生物痰消化液，震荡保护剂为无硅消泡剂F1152，每1L安图生物痰消化液中加入的振荡保护剂的质量为0.1g。
实施例2
本实施例的痰液样本的消化方法，包括以下步骤：取痰液样本1mL，加1mL含震荡保护剂的复合痰消化液，然后在室温的环境中震荡10min。
本实施例采用的复合痰消化液是将震荡保护剂加入安图生物痰消化液中混合得到；采用的震荡保护剂为吐温-80，每1L安图生物痰消化液中加入的震荡保护剂的质量为1g。
实施例3
本实施例的痰液样本的消化方法与实施例1的区别仅在于：本实施例采用的震荡保护剂为无硅消泡剂F1152和吐温-80，每1L安图生物痰消化液中加入的无硅消泡剂F1152的质量为0.1g，每1L安图生物痰消化液中加入的吐温-80的质量为1g。
实施例4
本实施例的痰液样本的消化方法与实施例1的区别仅在于：本实施例采用的痰消化液为OXOID痰消化液，震荡保护剂为蔗糖，每1L的OXOID痰消化液中加入的振荡保护剂的质量为1g。
实施例5
本实施例的痰液样本的消化方法与实施例1的区别仅在于：本实施例采用的震荡保护剂为动物源蛋白胨，每1L安图生物痰消化液中加入的振荡保护剂的质量为5g。
实施例6～52
实施例6～52的痰液样本的消化方法，包括以下步骤：取痰液样本1mL，加1mL含震 荡保护剂的复合痰消化液，然后在室温的环境中震荡10min。所采用复合痰消化液是将震荡保护剂加入痰消化液中混合得到。具体各实施例中采用的痰消化液、震荡保护剂和震荡保护剂的用量见表1。
表1实施例6～52中采用痰消化液、震荡保护剂和震荡保护剂的用量
Figure PCTCN2021120824-appb-000001
Figure PCTCN2021120824-appb-000002
Figure PCTCN2021120824-appb-000003
实验例1
取52份1mL的痰液样本分别按照实施例1～52痰液样本的消化方法进行消化；另外 设置与52个实施例一一对应的52个对比例，各对比例与对应实施例的区别仅在省去所采用的震荡保护剂；分别取与对应实施例相同的痰液样本1mL按照各对比例的方法进行消化。
然后用移液器分别吸取每个消化完成的处理液10微升接种至对应培养基上(各对比例和对应实施例的培养基完全相同)，用接种环进行划线，最后放置35～37℃条件下进行细菌学培养，然后对各培养基随机选择的一种或几种菌(各对比例和对应实施例选取的菌一致)采用平板计数法统计各培养基中的活菌数，结果见表2。
表2各实施例及对应对比例的培养基中选取菌的活菌数统计结果
Figure PCTCN2021120824-appb-000004
Figure PCTCN2021120824-appb-000005
Figure PCTCN2021120824-appb-000006
Figure PCTCN2021120824-appb-000007
由表2中数据可知，添加震荡保护剂的痰消化液对痰样本中的菌有较好的保护作用。
实验例2
用新鲜活化的卡他莫拉菌ATCC25238制成0.5麦氏的菌悬液，从中取3份完全相同的菌悬液(每份1mL)，然后任取其中1份(第1份)加入1mL的生理盐水，再取1份(第2份)加入1mL安图生物痰消化液，最后1份(第3份)加入1mL复合痰消化液，得到3个样品。再重复上述过程两次，将后两次重复后得到的样品在室温的环境中震荡10min。其中复合痰消化液是将震荡保护剂加入安图生物痰消化液中混合得到；每1L安图生物痰消化液中加入的震荡保护剂的质量为1g；所采用的震荡保护剂为吐温-80。
每类样品震荡前后的形貌见图1，其中第1列的第1行为第一次得到的第1份菌悬液加入生理盐水未进行震荡的示意图，第1列的第2～3行为后两次重复时得到的第1份菌悬液加入生理盐水震荡后的示意图。第2列的第1行为第一次得到的第2份菌悬液加入安图生物痰消化液未进行震荡的示意图，第2列的第2～3行为后两次重复时得到的第2份菌悬液加入安图生物痰消化液震荡后的示意图。第3列的第1行为第一次得到的第3份菌悬液加入复合痰消化液未进行震荡的示意图，第3列的第2～3行为后两次重复时得到的第3份菌悬液加入复合痰消化液震荡后的示意图。由图1可知，是震荡本身导致菌落死亡，添加震荡保护剂的 安图生物痰消化液震荡后菌落明显多于不加震荡保护剂的安图生物痰消化液。
实验例3
用新鲜活化的化脓链球菌ATCC19615制成0.5麦氏的菌悬液，从中取3份完全相同的菌悬液(每份1mL)，然后任取其中1份(第1份)加入1mL的复合痰消化液，再取1份(第2份)加入1mL的生理盐水，最后1份(第3份)加入1mL的安图生物痰消化液，得到3个样品。再重复上述过程两次，将后两次重复后得到的样品在室温的环境中震荡10min。其中复合痰消化液是将震荡保护剂加入安图生物痰消化液中混合得到，每1L安图生物痰消化液中加入的震荡保护剂的质量为1g；所采用的震荡保护剂为吐温-80。
每类样品震荡前后的形貌见图2，其中第1列的第1行为第一次得到的第1份菌悬液加入复合痰消化液未进行震荡的示意图，第1列的第2～3行为后两次重复时得到的第1份菌悬液加入复合痰消化液震荡后的示意图。其中第2列的第1行为第一次得到的第2份菌悬液加入生理盐水未进行震荡的示意图，第2列的第2～3行为后两次重复时得到的第2份菌悬液加入生理盐水震荡后的示意图。第3列的第1行为第一次得到的第3份菌悬液加入安图生物痰消化液未进行震荡的示意图，第3列的第2～3行为后两次重复时得到的第3份菌悬液加入安图生物痰消化液震荡后的示意图。由图2可知，是震荡本身导致菌落死亡，添加震荡保护剂的安图生物痰消化液震荡后菌落明显多于不加震荡保护剂的安图生物痰消化液。
实验例4
用新鲜活化的口腔链球菌ATCC6429制成0.5麦氏的菌悬液，从中取3份完全相同的菌悬液(每份1mL)，然后任取其中1份(第1份)加入1mL的复合痰消化液，再取1份(第2份)加入1mL的生理盐水，最后1份(第3份)加入1mL的OXOID痰消化液，得到3个样品。再重复上述过程两次，将后两次重复后得到的样品在室温的环境中震荡10min。其中复合痰消化液是将震荡保护剂加入OXOID痰消化液中混合得到；所采用的震荡保护剂为吐温-80+无硅消泡剂F1152，每1L的OXOID消化液中加入的无硅消泡剂F1152的质量为0.1g，加入的吐温-80的质量为1g。
每类样品震荡前后的形貌见图3，其中第1列的第1行为第一次得到的第1份菌悬液加入复合痰消化液未进行震荡的示意图，第1列的第2～3行为后两次重复时得到的第1份菌悬液加入复合痰消化液震荡后的示意图。其中第2列的第1行为第一次得到的第2份菌悬液加入生理盐水未进行震荡的示意图，第2列的第2～3行为后两次重复时得到的第2份菌悬液加入生理盐水震荡后的示意图。第3列的第1行为第一次得到的第3份菌悬液加入OXOID痰消化液未进行震荡的示意图，第3列的第2～3行为后两次重复时得到的第3份菌悬液加入 OXOID痰消化液震荡后的示意图。由图3可知，是震荡本身导致菌落死亡，添加震荡保护剂的OXOID痰消化液震荡后菌落明显多于不加震荡保护剂的OXOID痰消化液。
实验例5
用新鲜活化的嗜麦芽窄食单胞菌ATCC17666制成0.5麦氏的菌悬液，从中取3份完全相同的菌悬液(每份1mL)，然后任取其中1份(第1份)加入1mL的复合痰消化液，再取1份(第2份)加入1mL的生理盐水，最后1份(第3份)加入1mL的OXOID痰消化液，得到3个样品。再重复上述过程两次，将后两次重复后得到的样品在室温的环境中震荡10min。其中复合痰消化液是将震荡保护剂加入OXOID痰消化液中混合得到；所采用的震荡保护剂为吐温-80+无硅消泡剂F1152，每1L的OXOID消化液中加入的无硅消泡剂F1152的质量为0.1g，加入的吐温-80的质量为的2g。
每类样品震荡前后的形貌见图4，其中第1列的第1行为第一次得到的第1份菌悬液加入复合痰消化液未进行震荡的示意图，第1列的第2～3行为后两次重复时得到的第1份菌悬液加入复合痰消化液震荡后的示意图。其中第2列的第1行为第一次得到的第2份菌悬液加入生理盐水未进行震荡的示意图，第2列的第2～3行为后两次重复时得到的第2份菌悬液加入生理盐水震荡后的示意图。第3列的第1行为第一次得到的第3份菌悬液加入OXOID痰消化液未进行震荡的示意图，第3列的第2～3行为后两次重复时得到的第3份菌悬液加入OXOID痰消化液震荡后的示意图。由图4可知，是震荡本身导致菌落死亡，添加震荡保护剂的OXOID痰消化液震荡后菌落明显多于不加震荡保护剂的OXOID痰消化液。

Claims (16)

  1. 一种痰液样本的消化方法，其特征在于：包括以下步骤：将痰液样本、痰消化液和震荡保护剂的混合物进行震荡消化；所述震荡保护剂选自消泡剂、表面活性剂、蛋白质、蛋白胨、多羟基有机物中的任意一种或组合。
  2. 根据权利要求1所述的痰液样本的消化方法，其特征在于：所述消泡剂为无硅消泡剂。
  3. 根据权利要求1所述的痰液样本的消化方法，其特征在于：所述表面活性剂为非离子表面活性剂。
  4. 根据权利要求3所述的痰液样本的消化方法，其特征在于：所述非离子表面活性剂选自聚氧乙烯型非离子表面活性剂、多元醇型非离子表面活性剂中的任意一种或组合。
  5. 根据权利要求4所述的痰液样本的消化方法，其特征在于：所述聚氧乙烯型非离子表面活性剂为聚乙二醇2000、聚乙二醇6000中的任意一种或组合。
  6. 根据权利要求4所述的痰液样本的消化方法，其特征在于：所述多元醇型非离子表面活性剂选自吐温-20、吐温-80中的一种或两种。
  7. 根据权利要求1所述的痰样本的消化方法，其特征在于：所述多羟基有机物为糖类物质。
  8. 根据权利要求7所述的痰液样本的消化方法，其特征在于：所述糖类物质选自糊精、海藻糖、蔗糖中的任意一种或组合。
  9. 根据权利要求1所述的痰液样本的消化方法，其特征在于：所述蛋白质为牛血清白蛋白、酪蛋白中的任意一种或组合。
  10. 根据权利要求1所述的痰液样本的消化方法，其特征在于：所述蛋白胨为动物源蛋白胨、植物源蛋白胨中的任意一种或组合。
  11. 根据权利要求1～10中任意一项所述的痰液样本的消化方法，其特征在于：每1L痰消化液对应使用的震荡保护剂的质量为0.01～41g。
  12. 根据权利要求1～10中任意一项所述的痰液样本的消化方法，其特征在于：所述震荡保护剂包括消泡剂；每1L痰消化液对应使用的消泡剂的质量为0.01～1g。
  13. 根据权利要求1～10中任意一项所述的痰液样本的消化方法，其特征在于：所述震荡保护剂包括表面活性剂；每1L痰消化液对应使用的表面活性剂的质量为0.1～10g。
  14. 根据权利要求1～10中任意一项所述的痰液样本的消化方法，其特征在于：所述震荡保护剂包括蛋白质；每1L痰消化液对应使用的蛋白质的质量为1～10g。
  15. 根据权利要求1～10中任意一项所述的痰液样本的消化方法，其特征在于：所述 震荡保护剂包括蛋白胨；每1L痰消化液对应使用的蛋白胨的质量为1～10g。
  16. 根据权利要求1～10中任意一项所述的痰液样本的消化方法，其特征在于：所述震荡保护剂包括多羟基有机物；每1L痰消化液对应使用的多羟基有机物的质量为1～10g。
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