TWI417388B - Method for regenerating and circulating adenosine triphosphate, method for detecting microorganisms in a sample and kit thereof - Google Patents

Description

Method for regenerating circulation of adenosine triphosphate, method for detecting microorganisms in sample and kit thereof

The present invention relates to a method for the regeneration cycle of adenosine triphosphate (ATP), and in particular to a method for regenerating adenosine triphosphate by using adenosine triphosphate regenerating enzyme in a luminescence reaction, and using this mechanism Detection of microbial content in the sample.

Since adenosine triphosphate (ATP) must be present in the organism, the microbial content in the sample can be estimated by detecting adenosine triphosphate in the sample. At present, the commonly used method is to convert the adenosine triphosphate reaction of the microorganism in the sample into cold light by cold light detection, and then use the luminescence spectrometer to measure the cold light emitted by the reaction, and the microbial content in the sample can be known. However, the disadvantage of this method is that it is greatly affected by human operation factors, and the detection sensitivity is not high. The currently known measurement methods or commercial products can detect microbial limits of about 10 ⁴ CFU/ml.

Microorganisms will gather together as they grow. In order to protect the ethnic group, the microorganisms secrete some substances, such as high molecular weight polymers such as polysaccharides, and coat themselves without external interference. These protective substances are also known as biofilms. Once the biofilm is formed, it is difficult to kill the microorganisms. Take Taiwan's high-tech industry as an example. Once the biofilm is found in the pipeline, the only way is to update the entire pipeline. If the water is clean, the growth of microorganisms in the water is slow, and there may be only a few bacteria in the beginning. However, with the current technology, it is impossible to detect that a biofilm has been formed when the microorganisms are found at the time of discovery. Therefore, there is a need for a highly sensitive method for detecting microorganisms.

In the current related research, WO0153513A1/(US2003064394) discloses that in a luminescence reaction, adenosine monophosphate (AMP) is converted into adenosine diphosphate (ADP) to regenerate adenosine triphosphate to enhance luminescence. . When there is only a small amount of adenosine triphosphate, adenosine monophosphate is converted to adenosine diphosphate by adenylate kinase. When a polyphosphoric acid compound is present, a phosphotransferase is used to convert adenosine monophosphate to adenosine diphosphate. The reaction temperature is between 30 and 50 ° C and the reaction time is about 10 to 100 minutes.

The present invention provides a method for adenosine triphosphate regeneration cycle comprising: providing adenosine triphosphate, luciferin and a luciferase for a luminescence reaction; and providing adenosine diphosphate-glucose (adenosine diphosphate-glucose, ADP-glucose) regenerates the enzyme with adenosine triphosphate and adds it to the luminescence reaction.

The invention also provides a method for detecting microorganisms in a sample, comprising: providing the same sample, the sample contains microorganisms; the sample is combined with a luminosin, a luminescent enzyme, an adenosine diphosphate-glucose and an adenosine triphosphate The regenerated enzyme is mixed and reacted to produce luminescence; and the intensity of the microorganisms in the sample is determined by the intensity of the luminescence.

The invention further provides a kit for the above method for detecting microorganisms in a sample, comprising: a luminescent pigment; a cold light enzyme, wherein the cold light leaven Catalyzing the adenosine triphosphate of the microorganism to react with the luminosin to produce luminescence and pyrophosphate (PPi); adenosine monophosphate (AMP); and an adenosine triphosphate regenerating enzyme, wherein The adenosine triphosphate regenerating enzyme catalyzes the reaction of the pyrophosphate with adenosine diphosphate-glucose to produce regenerated adenosine triphosphate, and the regenerated adenosine triphosphate reacts with the luciferin to enhance the luminescence intensity.

The above and other objects, features and advantages of the present invention will become more apparent from

The present invention relates to a method for adenosine triphosphate regeneration cycle, and which enhances the luminescence generated in a luminescence reaction system by adding an adenosine triphosphate regenerating enzyme, and the detailed mechanism is as follows.

See Figure 1. Adenosine triphosphate, luciferin and luciferase provide a luminescence reaction and produce luminescence, a well-known luminescence system. In an embodiment of the invention, adenosine triphosphate in the luminescence system can be regenerated to enhance luminescence.

First, adenosine triphosphate 101, luciferin 103, and luciferase 107 are provided for a cold light reaction. The concentration of adenosine triphosphate may be from about 0.1 nM to 10 nM, the concentration of luminescent photo 103 may be from about 1 μM to 100 μM, and the unit amount of cold photoenzyme may be from about 0.5 U to 10 U. Cold light enzyme 107 can catalyze the luminescence reaction to react adenosine triphosphate 101, luminescent photo103 and oxygen oxygen 105, and produce pyrophosphate (pyrophosphate, PPi) 109, adenosine monophosphate (AMP) 111, oxyluciferin 113 and carbon dioxide 115 products and luminescent light 117.

Further, adenosine diphosphate-glucose 119 and adenosine triphosphate regenerating enzyme 121 are supplied and added to the above luminescent reaction. The concentration of adenosine diphosphate-glucose 119 can range from about 0.5 [mu]M to 10 [mu]M, while the concentration of adenosine triphosphate regenerating enzyme 121 can range from about 0.4 mg/ml to 3 mg/ml. The adenosine triphosphate regenerating enzyme 121 may include ADP-gliuse pyrophosphorylase. Adenosine triphosphate regenerating enzyme 121 catalyzes the reaction of pyrophosphate 109 with adenosine diphosphate-glucose 119 to produce regenerated adenosine triphosphate 101 and glucose-1-phosphate 123.

It should be noted that the above-mentioned regenerated adenosine triphosphate 101 will enter the luminescence reaction to continue the generation of cold light and enhance the intensity of cold light, and also complete the cycle of adenosine triphosphate regeneration. In one embodiment, adenosine triphosphate 101, luciferin 103, luminescent enzyme 107, adenosine diphosphate-glucose 119, and adenosine triphosphate regenerating enzyme 121 are provided simultaneously.

Moreover, the method of the adenosine triphosphate regeneration cycle of the present invention is more conducive to the improvement of the cold light intensity of the lower concentration of adenosine triphosphate. In one embodiment, the lower concentration of adenosine triphosphate is from about 0.05 nM to 4 nM.

In one embodiment, the above method may further comprise providing a magnesium ion and/or a tris (hydroxymethyl) aminomethane, Tris buffer solution to assist in the reaction. Magnesium ions can be provided by, for example, magnesium chloride.

In still other embodiments, the above method may further comprise providing a magnesium ion, a Tris buffer solution, fructose-1, 6-bisphosphate (FBP), and/or glucose 2,6-phosphate via phosphorus. Phosphoglucomutase (PGM) to assist in the reaction.

According to the mechanism of the above method, the present invention can also provide a method for detecting microorganisms in a sample, which can detect the presence or absence of microorganisms in the sample, and simultaneously detect the microbial content in the sample in the presence of microorganisms in the sample.

The same is provided first, and the sample may contain microorganisms. The sample can include a liquid sample. In an embodiment, the sample may include drinking water, domestic water, industrial wastewater, water in a pure water system line, or water in an ultrapure water system line. In an embodiment, the sample may be pre-processed. The pretreatment can, for example, concentrate the original sample, heat the sample for about 5-30 minutes, and the like.

The sample is then mixed with luciferin, luminescent enzyme, adenosine diphosphate-glucose and adenosine triphosphate regenerating enzyme for a reaction. The reaction time is from about 90 seconds to 10 minutes, preferably 10 minutes. The temperature of the reaction is room temperature, in one embodiment about 15-30 °C.

Referring to Fig. 2, if the sample contains microorganisms, in the above reaction, the luminescent enzyme catalyzes the reaction of the adenosine triphosphate of the microorganism with the luminosity 201 to produce pyrophosphate 203, adenosine monophosphate 205, oxidized luminescent 207 and carbon dioxide. The product of 209 and cold light 211. In addition, adenosine triphosphate regenerating enzyme 213 catalyzes the reaction of pyrophosphate 203 with adenosine diphosphate-glucose 215 to produce regenerated adenosine triphosphate 217 and phosphoglucose 219, and the regenerated adenosine triphosphate 217 will enter cold light again. In reaction 201, adenosine triphosphate is regenerated and the cold light intensity is enhanced. In one embodiment, the desired sample volume for performing the reaction is from about 5 to about 50 μl, preferably 10 μl. The concentration of luciferin may be about 50 μM-400 μM, the unit amount of luminescent enzyme may be about 0.5 U-10 U, the concentration of adenosine diphosphate-glucose 215 may be about 1 μM-100 μM, and the concentration of adenosine triphosphate regenerating enzyme 213 may be Is about 0.4mg/ml-3 Mg/ml. Further, the adenosine triphosphate regenerating enzyme may include adenosine diphosphate-glucose pyrophosphatase.

In still another embodiment, a magnesium ion and/or a Tris buffer solution may be further provided in the above reaction to assist the reaction. Magnesium ions can be provided by, for example, magnesium chloride.

In still other embodiments, a magnesium ion, a Tris buffer solution, fructose (Fructose-1, 6-bisphosphate, FBP), and/or 2,6-phosphate glucose may be displaced by glucose phosphate. A phosphoglucomutase (PGM) is added to the above reaction to assist the reaction.

Finally, by determining the content of microorganisms in the sample by the intensity of cold light, the stronger the intensity of cold light, the higher the microbial content in the sample. The method of the present invention for detecting microorganisms in a sample helps to increase the luminescence intensity when the sample microbial content is low. In one embodiment, in the method for detecting microorganisms in a sample of the present invention, the microbial content has a linear relationship with the intensity of the luminescence. In still another embodiment, a standard curve of the relative relationship between the intensity of the cold light and the microbial content may be first prepared, and according to the standard curve, the microbial content in the sample is obtained by the intensity of the cold light measured by the sample. The method for detecting microorganisms in the sample can detect that the microbial content in the sample ranges from 10 ^{3 to} 10 ⁸ CFU/ml, preferably the microbial content in the detectable sample is less than 10 ³ CFU/ml.

In another aspect, the invention can also provide a kit for use in the method of the invention for detecting microorganisms in a sample.

The kit may include a luciferin, a luminescent enzyme, an adenosine diphosphate-glucose, and an adenosine triphosphate regenerating enzyme. The cold light enzyme catalyzes the reaction of microbial adenosine triphosphate with luciferin to produce luminescence and pyrophosphate, while the adenosine triphosphate regenerating enzyme catalyzes the reaction of pyrophosphate with adenosine diphosphate-glucose to produce regenerated adenosine triphosphate. Regenerated adenosine triphosphate and luciferin React to enhance the intensity of cold light.

The concentration of luminescent light in the above set may be about 50 μM-400 μM, the unit amount of cold light enzyme may be about 0.5 U-10 U, and the concentration of adenosine diphosphate-glucose may be about 1 μM-100 μM, adenosine triphosphate regenerating enzyme. The concentration can range from about 0.4 mg/ml to 3 mg/ml. Further, the adenosine triphosphate regenerating enzyme may include adenosine diphosphate-glucose pyrophosphatase.

In one embodiment, the kit may further comprise a magnesium ion and/or Tris buffer solution to aid in the reaction. Magnesium ions can be provided by, for example, magnesium chloride.

In still other embodiments, the kit may further comprise a magnesium ion, a Tris buffer solution, fructose-1, 6-bisphosphate (FBP), and/or 2,6-phosphate glucose via glucose phosphate. Phosphoglucomutase (PGM) is used to assist the reaction.

[Examples]

Example 1

Comparison of the adenosine triphosphate regeneration cycle method and the cold light intensity produced by this method in the absence of adenosine diphosphate-glucose

The reaction materials required for the luminescence reaction were mixed together, and adenosine diphosphate-glucose (experimental group) and no adenosine diphosphate-glucose (control group) were added, respectively. After 90 seconds of reaction, the intensity of the cold light was then detected. The reaction conditions are shown in Table 1, and the results are shown in Fig. 3.

Fig. 3 shows that the amount of cold light intensity obtained by the experimental group having the reaction starting material adenosine diphosphate-glucose was much higher than that of the control group lacking the reaction adenosine diphosphate-glucose, which was about 4 times. Thus, the experimental group showing the reaction starting material adenosine diphosphate-glucose was shown to contribute to the improvement of the intensity of cold light.

Example 2

Effect of initial concentration of adenosine triphosphate on cold light intensity

1. The initial concentration of adenosine triphosphate is 30nM

Mixing the reaction materials required for the luminescence reaction into separate additions Adenosine diphosphate-glucose (experimental group) was followed by no adenosine diphosphate-glucose (control group) and only adenosine diphosphate-glucose pyrophosphatase (control group). The above three groups were reacted for 10 minutes and then respectively detected for cold light intensity. The reaction conditions are shown in Table 2, and the results show Fig. 4a.

2. The initial concentration of adenosine triphosphate is 1nM

The reaction materials required for the luminescence reaction were mixed together and divided into adenosine diphosphate-glucose (experimental group) without adenosine diphosphate-glucose (control group) and adenosine diphosphate-glucose pyrophosphatase only. (Control group) three groups. The above three groups were reacted for 10 minutes and then respectively detected for cold light intensity. The reaction conditions are shown in Table 3, and the results show Figure 4b.

Figure 4a shows the intensity of the cold light produced by the adenosine triphosphate regeneration cycle and the general luminescence reaction at an initial concentration of adenosine triphosphate of 30 nM. Figure 4b shows the intensity of the cold light produced by the adenosine triphosphate regeneration cycle method and the general luminescence reaction at an initial concentration of adenosine triphosphate of 1 nM.

Figure 4a shows that when the initial concentration of adenosine triphosphate is 30 nM, the adenosine triphosphate regeneration cycle produces a luminescence intensity of about 1.4 times the intensity of the cold light produced by a typical luminescence reaction at 30 minutes, and 4b. The graph shows that when the initial concentration of adenosine triphosphate is 1 nM, the adenosine triphosphate regeneration cycle produces a luminescence intensity of about 3.6 times that of a typical luminescence reaction when the reaction is carried out for 30 minutes. Therefore, it can be known that the method of the adenosine triphosphate regeneration cycle of the present invention is more helpful to initiate the increase of the cold light intensity of the lower concentration of adenosine triphosphate.

Example 3

The method for detecting microorganisms in a sample of the invention and the comparison of the luminescence intensity produced by the microorganisms in the sample without adenosine diphosphate-glucose

1. Escherichia coli BL21

The reaction materials required for the luminescence reaction were mixed together, and the bacterial liquid (10 ³ -10 ⁵ CFU/ml) after boiling for 15 minutes at a high temperature was added, and then adenosine diphosphate-glucose (experimental group) was added separately and not added. Adenosine diphosphate-glucose (control). After 10 minutes of reaction, the intensity of the cold light was detected. The detailed reaction conditions are shown in Table 4, and the results of the experimental group and the control group are shown in Figure 5a.

2. Pseudomonas aeruginosa PAO1

The reaction materials required for the luminescence reaction were mixed together, and the bacterial liquid (10 ³ -10 ⁵ CFU/ml) after boiling for 15 minutes at a high temperature was added, and then adenosine diphosphate-glucose (experimental group) was added separately and not added. Adenosine diphosphate-glucose (control). After 10 minutes of reaction, the intensity of the cold light was detected. The detailed reaction conditions are shown in Table 5. The results of the experimental group and the control group are shown in Figure 5b.

Table 5, reaction conditions

From Fig. 5a and Fig. 5b, it can be seen that the amount of cold light intensity obtained by the experimental group with adenosine diphosphate-glucose is higher than that of the lack of the reaction starting material adenosine diphosphate-glucose at all bacteria-containing levels. Group height.

Example 4

Comparison of the method for detecting microorganisms in a sample and the intensity of cold light produced by a general cold light reaction

1. Use a commercial package

The cold light intensity produced by Escherichia coli DH5α ( Escherichia coli DH5α) of 10 ⁴ -10 ⁸ CFU/ml was measured by a commercial kit (AMSALite III ^TM , AMSA, Inc. (Antimicrobial Specialists & Associates, Inc.). In Figure 6.

2. Use the kit of the invention

See Example 3: Experiment of the experimental group portion of E. coli BL21, the results of which are shown in Figure 5a.

It can be seen from Fig. 6 that when the amount of Escherichia coli is less than 10 ⁴ CFU/ml, cold light cannot be detected. From Fig. 5a, it can be seen that when the amount of Escherichia coli is 10 ³ CFU/ml, cold light can still be detected. Therefore, the results show that the sensitivity of the method for detecting microorganisms in a sample of the present invention is higher than that of a general cold light reaction.

Example 5

Relationship between microbial content and cold light intensity of the method for detecting microorganisms in a sample of the invention

1. Escherichia coli BL21

Mixing the reaction materials required for the luminescence reaction, adding 10 ³ -10 ⁵ CFU/ml of the bacterial solution after boiling for 15 minutes at high temperature, and adding adenosine diphosphate-glucose (experimental group) separately, and Adenosine diphosphate-glucose was added as a control group. After 10 minutes of reaction, the luminescence intensity produced by the samples containing different bacteria contents was detected separately. The detailed reaction conditions are shown in Table 6, and the experimental group results are shown in Figure 7.

Table 6, reaction conditions

Figure 7 shows that the samples with E. coli BL21 content of 10 ³ CFU/ml, 10 ⁴ CFU/ml and 10 ⁵ CFU/ml produced cold light intensities of 14.89 RLU, 53.97 RLU and 86.18 RLU, respectively. That is to say, when the method for detecting microorganisms in the sample is used, when the content of Escherichia coli BL21 is increased, the intensity of cold light generated is also enhanced, and the content of BL21 in E. coli is linear with the intensity of cold light, and the equation is y= 35.645x-19.61, R ² =0.9969.

2. Pseudomonas aeruginosa PAO1

Mixing the reaction materials required for the luminescence reaction, adding 10 ³ -10 ⁵ CFU/ml of the bacterial solution after boiling for 15 minutes at high temperature, and adding adenosine diphosphate-glucose (experimental group) separately, and Adenosine diphosphate-glucose was added as a control group. After 10 minutes of reaction, the luminescence intensity produced by the samples containing different bacteria contents was detected separately. Detailed reaction conditions are shown in Table 7, and experimental group results are shown in Figure 8.

Figure 8 shows that the P. aeruginosa PAO1 content of 10 ³ CFU/ml, 10 ⁴ CFU/ml and 10 ⁵ CFU/ml samples produced a luminescence intensity of 15.81 RLU, 33.53 RLU and 60.1 RLU, respectively. That is to say, when the method for detecting microorganisms in the sample is used, when the content of Pseudomonas aeruginosa PAO1 is increased, the intensity of cold light generated is also enhanced, and the content of PAO1 of Pseudomonas aeruginosa is linear with the intensity of cold light, and the equation is y=22.145x-7.81, R ² =0.9869.

3. Cactus ( Bacillus cereus )

Mixing the reaction materials required for the luminescence reaction, adding 10 ³ -10 ⁵ CFU/ml of the bacterial solution after boiling for 15 minutes at high temperature, and adding adenosine diphosphate-glucose (experimental group) separately, and Adenosine diphosphate-glucose was added as a control group. After 10 minutes of reaction, the luminescence intensity produced by the samples containing different bacteria contents was detected separately. The detailed reaction conditions are shown in Table 8, and the experimental group results are shown in Figure 9.

Figure 9 shows samples with a Cactus bacillus content of 10 ³ CFU/ml, 10 ⁴ CFU/ml and 10 ⁵ CFU/ml, which produced a luminescence intensity of 15.74 RLU, 47.98 RLU and 88.51 RLU, respectively. That is to say, using the method of the present invention for detecting microorganisms in a sample, when the content of the cactus bacillus is increased, the intensity of the luminescence generated is also enhanced, and the content of the cactus bacillus is linear with the luminescence intensity, and the equation is y=36.385x. -22.027, R ² = 0.9957.

The above experimental results show that the biological samples of different microorganisms are tested by the method for detecting microorganisms in the sample according to the present invention, and the cold light intensity produced by different kinds of microorganisms is enhanced with the increase of the microbial content, and the microbial content and the cold light intensity are enhanced. They also all exhibit a linear relationship. Therefore, from this result, it can be known that the method of detecting microorganisms in a sample of the present invention can detect the intensity of cold light of an unknown sample, and thereby estimate the microbial content in the sample.

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

101. . . Adenosine triphosphate

103. . . Luciferin

105. . . oxygen

107. . . Cold light enzyme

109. . . Pyrophosphate

111. . . Adenosine monophosphate

113. . . Oxidized luminescent phototin

115. . . carbon dioxide

117. . . Cold light

119. . . Adenosine diphosphate-glucose

121. . . Adenosine triphosphate regenerating enzyme

123. . . Phosphoric acid-glucose

201. . . Cold light enzyme catalyzes the reaction of microbial adenosine triphosphate with luciferin

203. . . Pyrophosphate

205. . . Adenosine monophosphate

207. . . Oxidized luminescent phototin

209. . . carbon dioxide

211. . . Cold light

213. . . Adenosine triphosphate regenerating enzyme

215. . . Adenosine diphosphate-glucose

217. . . Regenerated adenosine triphosphate

219. . . Phosphoric acid-glucose

Figure 1 shows the reaction mechanism of the method of the adenosine triphosphate regeneration cycle of the present invention.

Figure 2 shows the method of the present invention for detecting microorganisms in a sample.

Figure 3 shows the adenosine triphosphate regeneration cycle method and the cold light intensity produced by this method in the absence of adenosine diphosphate-glucose.

Figure 4a shows the intensity of the cold light produced by the adenosine triphosphate regeneration cycle and the general luminescence reaction at an initial concentration of adenosine triphosphate of 30 nM.

Figure 4b shows the intensity of the cold light produced by the adenosine triphosphate regeneration cycle method and the general luminescence reaction at an initial concentration of adenosine triphosphate of 1 nM.

Figure 5a shows the method of the present invention for detecting microorganisms in a sample and the cold light intensity produced by the method of E. coli BL21 in the absence of adenosine diphosphate-glucose.

Figure 5b shows the method of detecting microorganisms in a sample of the present invention and the cold light intensity produced by Pseudomonas aeruginosa PAO1 in the sample without adenosine diphosphate-glucose.

Figure 6 shows the intensity of the luminescence produced by a typical luminescence reaction.

Fig. 7 is a graph showing the relationship between the increase in the content of Escherichia coli BL21 and the detected intensity of cold light using the method of the present invention for detecting microorganisms in a sample.

Figure 8 is a graph showing the relationship between the increase in the content of Pseudomonas aeruginosa PAO1 and the detected intensity of cold light using the method of the present invention for detecting microorganisms in a sample.

Figure 9 is a graph showing the relationship between the increase in the content of the cactus and the detected intensity of cold light using the method of the present invention for detecting microorganisms in a sample.

101. . . Adenosine triphosphate

103. . . Luciferin

105. . . oxygen

107. . . Cold light enzyme

109. . . Pyrophosphate

111. . . Adenosine monophosphate

113. . . Oxidized luminescent phototin

115. . . carbon dioxide

117. . . Cold light

119. . . Adenosine diphosphate-glucose

121. . . Adenosine triphosphate regenerating enzyme

123. . . Phosphoric acid-glucose

Claims (35)

A method for regenerating adenosine triphosphate, comprising: providing adenosine triphosphate, a luminescent acid and a luminescent enzyme for performing a luminescence reaction; and providing an adenosine diphosphate-glucose and an adenosine diphosphate-glucose coke Phosphatase is added to the luminescence reaction. A method for regenerating adenosine triphosphate as described in claim 1, wherein the adenosine triphosphate, the luciferin, the luminescent enzyme, the adenosine diphosphate-glucose, and the adenosine diphosphate-glucose coke Phosphatase is provided simultaneously. The method of regenerating the adenosine triphosphate according to claim 1, wherein the concentration of the adenosine triphosphate is from about 0.1 nM to 10 nM. The method of the adenosine triphosphate regeneration cycle of claim 1, wherein the concentration of the luciferin is from about 1 μM to 100 μM. The method of regenerating the adenosine triphosphate according to claim 1, wherein the unit amount of the luminescent enzyme is about 0.5 U to 10 U. The method of the adenosine triphosphate regeneration cycle of claim 1, wherein the adenosine diphosphate-glucose concentration is from about 0.5 μM to 10 μM. The method of the adenosine triphosphate regeneration cycle of claim 1, wherein the adenosine diphosphate-glucose pyrophosphatase concentration is about 0.4 mg/ml to 3 mg/ml. The method of the adenosine triphosphate regeneration cycle of claim 1, further comprising providing a magnesium ion. A method of regenerating adenosine triphosphate as described in claim 8 wherein the magnesium ion is provided by magnesium chloride. The method of the adenosine triphosphate regeneration cycle of claim 1, further comprising providing a Tris buffer solution. A method for detecting microorganisms in a sample, comprising: providing the same; mixing the sample with a luminosin, a luminescent enzyme, an adenosine diphosphate-glucose and an adenosine diphosphate-glucose pyrophosphatase, and performing a reaction And producing cold light; and determining the content of microorganisms in the sample by the intensity of cold light. The method for detecting microorganisms in a sample according to claim 11, further comprising performing a pretreatment on the sample, wherein the pretreatment comprises concentration or heating. The method of detecting microorganisms in a sample according to claim 12, wherein the heating is to boil the sample for about 5-30 minutes. The method for detecting microorganisms in a sample according to claim 11, wherein the microbial content in the sample is linear with the intensity of the cold light. The method of detecting microorganisms in a sample according to claim 11, wherein the sample may include a liquid sample. The method for detecting microorganisms in a sample according to claim 11, wherein the sample comprises drinking water, domestic water, industrial wastewater, water in a pure water system pipeline, or water in an ultrapure water system pipeline. The method for detecting microorganisms in a sample according to claim 11, wherein the volume of the sample required to carry out the reaction is about 5 to 50 μl. The method for detecting microorganisms in a sample according to the invention of claim 11, wherein the concentration of the luciferin is from about 1 μM to 100 μM. The method for detecting microorganisms in a sample according to claim 11, wherein the unit amount of the luminescent enzyme is about 0.5 U to 10 U. The method for detecting microorganisms in a sample according to claim 11, wherein the adenosine diphosphate-glucose concentration is about 0.5 μM to 10 μM. The method for detecting microorganisms in a sample according to claim 11, wherein the concentration of the adenosine diphosphate-glucose pyrophosphatase is about 0.4 mg/ml to 3 mg/ml. The method for detecting microorganisms in a sample according to claim 11, wherein the reaction time is about 90 seconds to 10 minutes. The method for detecting microorganisms in a sample according to claim 11, wherein the temperature of the reaction is room temperature. The method for detecting microorganisms in a sample according to claim 11, wherein the method for detecting microorganisms in the sample detects a microorganism content ranging from 10 ^{3 to} 10 ⁸ CFU/ml. The method for detecting microorganisms in a sample according to claim 11 of the patent application, further comprising providing a magnesium ion in the reaction. A method for detecting microorganisms in a sample according to claim 25, wherein the magnesium ion is provided by magnesium chloride. The method for detecting microorganisms in a sample according to claim 11 of the patent application, further comprising providing a tris buffer solution in the reaction. A kit for detecting microorganisms in a sample, which is used to apply for a patent The method for measuring microorganisms in a sample according to Item 11, comprising: a luminescent pigment; a luminescent enzyme, wherein the luminescent enzyme catalyzes the reaction of the adenosine triphosphate of the microorganism with the luminescent luminescence to produce luminescence and pyrophosphate; a diphosphate diphosphate-glucose; and an adenosine diphosphate-glucose pyrophosphatase, wherein the adenosine diphosphate-glucose pyrophosphatase catalyzes the reaction of the pyrophosphate with adenosine diphosphate-glucose to produce regenerated adenosine triphosphate, And the regenerated adenosine triphosphate reacts with the luminescent luminescence to enhance the luminescence intensity. A kit for detecting microorganisms in a sample as described in claim 28, wherein the concentration of the luciferin is from about 1 μM to 100 μM. The kit for detecting microorganisms in the sample as described in claim 28, wherein the unit amount of the luminescent enzyme is about 0.5 U to 10 U. The set of microorganisms in the detection sample as described in claim 28, wherein the adenosine diphosphate-glucose concentration is about 0.5 μM to 10 μM. The kit for detecting microorganisms in the sample according to claim 28, wherein the concentration of the adenosine diphosphate-glucose pyrophosphatase is about 0.4 mg/ml to 3 mg/ml. The detection of the microbial set in the sample as described in claim 28 of the patent application further includes providing a magnesium ion. A kit for detecting microorganisms in a sample as described in claim 33, wherein the magnesium ion is provided by magnesium chloride. The kit for detecting microorganisms in the sample as described in claim 28 of the patent application includes a tris buffer solution.