阅读说明：本技术 一种基于丙酮酸激酶检测样品中微生物的方法及试剂盒 (Method and kit for detecting microorganisms in sample based on pyruvate kinase ) 是由 谈翼辰 李兴祥 程俊雨 秦爱清 林学祥 肖鑫 于 2019-10-10 设计创作，主要内容包括：本发明公开一种基于丙酮酸激酶检测样品中微生物的方法及试剂盒。所述检测方法包括靶微生物细胞的裂解处理,加入检测试剂,温育转化以及测定分析等步骤。所述试剂盒包括该检测方法所需的所有检测试剂及裂解缓冲液,包括含有通过丙酮酸激酶转化成ATP必需组分的第一试剂,和用于检测ATP的荧光素/荧光素酶系统的第二试剂,第一试剂和第二试剂以母液试剂形式提供。在实际操作中将母液试剂可以和裂解缓冲液加在一起使用。操作更加快捷灵敏准确具有优良的使用价值。(The invention discloses a method and a kit for detecting microorganisms in a sample based on pyruvate kinase. The detection method comprises the steps of target microorganism cell lysis treatment, detection reagent addition, incubation conversion, determination analysis and the like. The kit includes all the detection reagents and lysis buffers required for the detection method, including a first reagent containing components necessary for conversion to ATP by pyruvate kinase, and a second reagent for the luciferin/luciferase system for detecting ATP, the first and second reagents being provided as mother liquor reagents. In practice, the mother liquor reagent may be used together with the lysis buffer. The operation is faster, more sensitive and more accurate, and the method has excellent use value.)

1. A method for detecting a microorganism in a sample based on pyruvate kinase, comprising: the method comprises the following steps:

s1: and (3) cracking treatment: lysing the target microorganism in the sample by a suitable lysis treatment method to release intracellular material for contacting with the reagent;

s2: adding a reagent: adding a first reagent and a second reagent into the sample subjected to the cracking treatment in the step S1 to prepare a conversion mixed solution; the first reagent contains components necessary for conversion to ATP by pyruvate kinase, including PEP and ADP, but not pyruvate kinase itself; the second reagent contains a luciferin/luciferase system for detecting ATP;

s3: and (3) incubation conversion: incubating the conversion mixture of step S2 for several minutes at room temperature to convert ADP to ATP, which is labeled with the luciferin/luciferase system;

s4: determination and analysis: measuring the light signal emitted by the ATP converted and generated in the step S3 by using a photometer, recording relative light units, and determining the amount of the ATP converted and generated; and correlating the amount of ATP measured with the amount of pyruvate kinase to determine the presence and/or amount of the target microorganism in the sample.

2. The pyruvate kinase-based method for detecting microorganisms in a sample according to claim 1, wherein: further comprising a sample preparation step of treating the sample to remove the disturbing factor and adjust the concentration of the sample before the lysis treatment of the target microorganism in step S1; the method for removing interference comprises a filtering method or treatment by using a human cell specific lysis reagent, or a combination of the two.

3. The pyruvate kinase-based method for detecting microorganisms in a sample according to claim 1, wherein: in step S1, the lysis treatment is to treat the sample with a lysis buffer capable of lysing the target microorganism.

4. The pyruvate kinase-based method for detecting a microorganism in a sample according to claim 3, wherein: the lysis buffer was a mixture of 0.15% cationic detergent and 0.25% tertiary diamine surfactant.

5. The pyruvate kinase-based method for detecting microorganisms in a sample according to claim 1, wherein: in step S2, the ADP is purified commercially available ADP, and the concentration of the added sample is 0.001 mM-10 mM; the purification method comprises column chromatography or the use of ATP-specific degrading enzymes, or a combination of the two methods.

6. The pyruvate kinase-based method for detecting microorganisms in a sample according to claim 1, wherein: in step S2, the first reagent and the second reagent are mixed in advance and added to the sample to prepare a reaction mixture.

7. The pyruvate kinase-based method for detecting microorganisms in a sample according to claim 1, wherein: further comprising the step of distinguishing the target microbial cell from a fungal spore by a control assay or by a double assay; the control assay is a control using a non-ionic detergent that lyses only fungal spores and somatic cells of the animal; the double detection is that ATP degrading enzyme is used between two detection cycles of adding non-ionic detergent capable of only cracking fungal spores and animal somatic cells and cracking buffering in a sample.

8. A kit for detecting microorganisms in a sample based on pyruvate kinase, comprising: the kit comprises a first reagent, a second reagent and a lysis buffer which are required by the pyruvate kinase-based method for detecting the microorganism in the sample according to the claims 1-9; the first reagent contains ADP, PEP and Mg necessary for conversion to ATP by pyruvate kinase²⁺The second reagent contains a luciferin/luciferase system for detecting ATP, and the first and second reagents are mixed and provided as a seed stock.

9. The pyruvate kinase-based kit for detecting a microorganism in a sample according to claim 8, wherein: the pH value of the mother liquor is 7.0-8.0, and the mother liquor is subpackaged in small bottles and is freeze-dried and stored at the temperature of-20 ℃.

10. The pyruvate kinase-based kit for detecting a microorganism in a sample according to claim 8, wherein: the lysis buffer and the mother liquor reagent may be mixed first and then added to the sample.

Technical Field

The invention relates to a method and a kit for detecting microorganisms in a sample, in particular to a method and a kit for detecting microorganisms in a sample based on pyruvate kinase.

Technical Field

Enzymatic assays determine the catalytic activity of an enzyme by measuring the substrate consumed by the enzyme or the product produced by the enzyme catalysis. Typical enzymatic assays include colorimetric assays and chemiluminescent assays using chemiluminescent substrates. Among them, the colorimetric analysis method is widely used, and the common colorimetric analysis methods include MTT analysis method, X gal assay method, and the like. The MTT assay is an assay for redox using a tetrazolium dye as a substrate; the X gal assay measures β -galactosidase activity using X-gal (5-bromo-4-chloro-3-indolyl- β -D-galactoside) as a substrate; the fluorometric method is a method of measuring a fluorescence value to calculate the content or activity of a corresponding substance, for example, measuring the activity of beta-galactosidase using 4-methylumbelliferyl-beta-D-galactoside. There are also variations on these assays, such as enzyme-coupled assays and fluorescence quenching assays or fluorescence energy transfer assays.

Enzyme assays are widely used to identify microorganisms, i.e., the presence of a target microorganism can be determined by detecting the presence of certain enzyme activities. Enzyme assays that detect specific enzymes have many applications, such as disease diagnosis, environmental monitoring, and drug discovery. Since the mid-twentieth sixties, one has begun to use ATP (adenosine triphosphate as an indicator of microbial numbers. since almost all organisms utilize ATP as a chemical energy source, ATP can be measured by ATP-driven luciferase or luciferin, i.e., the light generated by the enzyme reaction can be measured by a photometric instrument and correlated with the amount of ATP present to determine the number of microbes in a sample^-12And (3) the ATP can be analyzed in a simple sample for several minutes, and the sample is generally only half an hour even if the sample is complex. However, this method has a significant disadvantage, ATP capacityThe ATP-enriched reagent is easy to be consumed by cell metabolism, and the content of the ATP is highly changed, so that the ATP content is detected only, the relationship between the ATP content and the number of related cells is not close enough, and the sensitivity is not enough in many cases. Thus, there remains a need for a method of detecting microorganisms or their contents in a sample that maintains the advantages of rapidity, is easy to operate, and is more sensitive.

It has been found that the speed and sensitivity of the ATP-based method can be greatly improved by simply replacing the target of the assay with ATP for the enzyme that produces it. Pyruvate kinase is an enzyme used by many organisms to convert Adenosine Diphosphate (ADP) to Adenosine Triphosphate (ATP), and the target substrate for this enzyme is preferably ADP. And pyruvate kinase is involved in the glycolytic pathway. It transfers the high-energy phosphate group of phosphoenolpyruvate (PEP) molecule to ADP to generate one molecule of ATP and one molecule of pyruvate, which requires Mg²⁺And the reaction is irreversible. Thus, pyruvate kinase activity is regulated by its own substrate PEP and the glycolytic intermediate fructose 1, 6-diphosphate, both of which increase pyruvate kinase activity. ATP, acetyl-CoA and alanine are allosteric inhibitors of the enzyme, which modulate the response. In view of the above, the present invention provides a method for detecting the presence of a target microorganism in a sample based on pyruvate kinase, and provides a kit for rapidly performing the detection process.

Disclosure of Invention

The present invention is directed to the aforementioned problems of the prior art ATP detection methods, and it is an object of the present invention to provide a method for detecting microorganisms or their contents in a sample that is fast, easy to operate, and more sensitive. In order to achieve the above purpose, the present invention provides a method and a kit for detecting microorganisms in a sample based on pyruvate kinase. The specific technical scheme is as follows:

the invention firstly provides a method for detecting microorganisms in a sample based on pyruvate kinase, which comprises the following steps:

s1: and (3) cracking treatment: the target microorganism in the sample is lysed by a suitable lysis treatment to release intracellular material for sufficient contact with the reagent.

S2: adding a reagent: to the sample subjected to the cleavage treatment in step S1, a first reagent containing a component necessary for conversion into ATP by pyruvate kinase and a second reagent containing a luciferin/luciferase system for detecting ATP are added to prepare a conversion mixture. The essential components of the first reagent which are converted into ATP by pyruvate kinase include PEP, ADP and the like, but do not include pyruvate kinase itself.

S3: and (3) incubation conversion: the conversion mixture described in step S2 is incubated at room temperature (15-30 ℃) for several minutes, the added ADP is converted to ATP by pyruvate kinase released by lysis of the sample target microorganism, and the ATP is labeled by the luciferin/luciferase system in the first reagent.

S4: determination and analysis: measuring the light signal emitted by the ATP converted in the step S3 by using a photometer, recording relative light units, and determining the amount of ATP; and correlating the amount of ATP measured with the amount of pyruvate kinase to determine the presence and/or quantity of the target microorganism in the sample.

As a preferred technical scheme, the method for detecting microorganisms in a sample based on pyruvate kinase further comprises sample preparation work, wherein before target microorganisms are lysed, the sample is treated by adopting a proper method so as to remove factors which interfere with the determination result in the sample. The method for removing the interference includes a filtration method or a method for removing the interfering substance in the sample using a somatic cell-specific lysis reagent, and the two methods are preferably used in combination.

In a preferred embodiment, in step S1, the lysis treatment is to treat the sample with a lysis buffer capable of lysing the target microorganism; the lysis buffer included commercially available cationic detergents and proprietary reagents.

Preferably, in step S2, the ADP in the first reagent is commercially available high-purity ADP with a purity of > 99.5%, and the ADP needs to be further purified before use to remove ATP contaminants therein; the further purification method comprises using column chromatography or using ATP specific degrading enzymes, or a combination of both methods.

Preferably, the ADP concentration in the sample is between 0.001mM and 10 mM. Preferably more than 0.005mM, more preferably more than 0.01mM, most preferably more than 0.05 mM. A particularly preferred amount of ADP is 0.1 mM. ADP and Mg in practical operation²⁺The upper limit of (b) can be determined experimentally by using a known number of bacteria.

Preferably, in step S2, the first reagent further contains fructose 1, 6-diphosphate capable of regulating pyruvate kinase, and magnesium ions necessary for the reaction, so as to ensure that ADP is carried out under sufficient magnesium ions; in addition, the first reagent further comprises acetyl-CoA, an allosteric inhibitor of pyruvate kinase, for regulating the reaction. The second reagent comprises luciferase, D-luciferin and MgCl₂Bovine serum albumin and a buffer solution; the pH value of the buffer solution is 5.5-8.5; the ideal pH value is 6-8, and the preferable pH value is about 7.5. Suitable buffers include Tris and phosphate buffers, which may also be used to collect and/or dilute samples in order to practice the present invention.

Preferably, the first reagent and the second reagent are mixed in advance, and then mixed with the treated sample, and the incubated conversion reaction and labeled ATP are directly completed in one step, and then the assay is performed.

As a preferred technical scheme, the method for detecting the microorganism in the sample based on the pyruvate kinase further comprises the steps of distinguishing target microorganism cells from fungal spores, distinguishing the target cells from the cells such as the fungal spores, and using a non-ionic detergent which can only crack the fungal spores and the somatic cells of the animal body as a control group, or adding the non-ionic detergent which can only crack the fungal spores and the somatic cells of the animal body before cracking the target microorganism in the product.

The invention also provides a kit for detecting microorganisms in a sample based on pyruvate kinase, which comprises a first reagent, a second reagent and a lysis buffer solution required by the method for detecting the microorganisms in the sample based on pyruvate kinase, wherein the first reagent contains a general reagentADP, PEP and Mg necessary for the conversion of perpyruvate kinase to ATP²⁺The second reagent contains a luciferin/luciferase system that detects ATP, and the first and second reagents are provided as a single mother liquor reagent.

As a preferred technical scheme, the mother liquor reagent specifically comprises the following components: 200mg of isopyrazole, 100mg mg of BSA, 2mg of fluorescein, 100mg of DTT, 100mg of acetyl-CoA, 220 MgCl 220 mg, 220 CaCl 220 mg, 2mg of luciferase, 10mg of gentamicin, 20mg of PEP, 2mg of fructose 1, 6-diphosphate, 5mg of ADP, and deionized water for preparing the system.

As a further preferred technical scheme, the lysis buffer and the mother liquor reagent can be mixed firstly and then added into the sample in one time, and the measurement and analysis can be directly carried out after the incubation.

The invention has the beneficial effects that:

compared with the existing ATP-based method for detecting microorganisms in samples, the method disclosed by the invention not only has the advantages of rapidness and sensitivity of an ATP detection method, but also has a more accurate detection result. In the ATP detection method, the ATP content is highly changed due to the consumption of ATP by cell metabolism, so that the detection result is not closely related to the number of related cells. The method of the invention relates the detected ATP amount to the amount of pyruvate kinase which is closely related to the number of target microorganisms or related cells in the sample, and the result is more reliable, thereby making up the important defects of the existing ATP-based method. In addition, the kit provided by the invention can quickly and effectively complete detection, is quicker, more sensitive and more accurate in operation than the existing ATP-based method, and has excellent use value.

Drawings

FIG. 1 is a calibration curve of the optical test for detecting Escherichia coli according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments.