CN112255209B

CN112255209B - Method for detecting microorganisms in fuel oil by using bioluminescence method and special detection rod thereof

Info

Publication number: CN112255209B
Application number: CN202011083184.4A
Authority: CN
Inventors: 刘龙飞
Original assignee: Beijing Zhongjian Baotai Biotechnology Co ltd
Current assignee: Beijing Zhongjian Baotai Biotechnology Co ltd
Priority date: 2020-10-12
Filing date: 2020-10-12
Publication date: 2021-09-14
Anticipated expiration: 2040-10-12
Also published as: CN112255209A

Abstract

The invention provides a method for detecting microorganisms in fuel oil by a bioluminescence method and a special detection rod thereof. The detection method of the invention adopts the mixture of apyrase, adenosine phosphate deaminase and copper sulfate as the free ATP remover, which can effectively remove the free ATP in the sample; octadecyl trimethyl ammonium chloride is adopted as a cell cracking agent, the cracking speed is high, the cracking is complete, and the residual ATP hydrolase can be irreversibly inactivated; the luciferase is freeze-dried powder and can be stored at normal temperature, so that the storage cost is saved; the rod part of the special matching detection rod adopts a thread design, so that the stability and consistency of results are ensured; the detection tube contains a fluorescence aggregation reaction ball for aggregating fluorescence signals, and the sensitivity, stability and accuracy of detection are improved.

Description

Method for detecting microorganisms in fuel oil by using bioluminescence method and special detection rod thereof

Technical Field

The invention relates to a preparation method and a determination method of a detection rod for rapidly detecting microorganisms in a fuel oil sample by an ATP bioluminescence method, belonging to the field of biochemical analysis and detection.

Background

Statistically, 50% of engine failures are caused by poor cleanliness of the fuel. Microorganisms are an important factor affecting fuel cleanliness, and have a significant impact on fuel storage and operational safety. Since 1930, the problem of microbial contamination of fuels has begun to be of concern given the growing and reproducing hazards of microorganisms in fuels. Microbial contamination is a main threat in the aviation industry, the marine industry and the transportation industry, a large amount of microbes and microbial secretions are condensed into viscous clusters or floccules which can block oil filters, oil pumps, fuel regulators and other accessories, the normal oil supply of an engine is directly influenced, and the operation safety is influenced; the secretion generated by the microorganisms can corrode a machine structure, damage a surface protection layer and sealant of an aluminum alloy structure of the oil tank, and once the protection coating is damaged, the base metal can be further corroded to penetrate through a wall plate of the oil tank, so that the oil tank leaks; microbial contamination can also lead to malfunction of the fuel level indicating device and inaccurate indicator readings. Therefore, it is necessary to control the microbial contamination of the fuel, and the microbial detection of the fuel is an important means for controlling the microbial contamination.

The detection method of the microorganism in the fuel mainly comprises a colony culture method, an indirect detection method and an ATP bioluminescence method. The colony culture method has high accuracy, does not need expensive and complicated professional equipment, has low culture cost, but has complex operation, long detection time, usually needs 4 days, can not realize real-time monitoring, has higher requirement on operators and needs strict sterile environment; the indirect method is mainly used for indirectly reacting whether the fuel is polluted by microorganisms by detecting the change of fuel performance indexes, such as appearance, copper sheet corrosion, solid particles, moisture, pH value, surface tension and the like, and the method has poor accuracy and low sensitivity; the ATP bioluminescence method determines the pollution condition of microorganisms by measuring the ATP content in the fuel, the ATP is the most main energy transfer molecule of all biological cells including bacteria and fungi, and the ATP content is in certain proportion to the viable bacteria amount, so the ATP is a reliable index for determining the microbial pollution degree of the fuel. The ATP method has high accuracy and sensitivity, is simple and quick to operate, can obtain results within 10 minutes, can realize the online detection of the oil tank and the oil storage tank, and is widely applied to airlines internationally.

The existing fuel microorganism detection pen adopting the ATP method has complex sample treatment and complex operation steps, can not directly detect ATP in microorganism cells, needs to respectively measure the total ATP content and the free ATP content, and the difference between the total ATP content and the free ATP content is ATP in the microorganism cells; meanwhile, the discrimination is poor; in addition, the existing fuel microorganism detection pen has high cost.

Based on the above situation, it is urgently needed to develop a detection method based on bioluminescence reaction, which can efficiently remove free ATP in a detection sample, effectively release ATP in microbial cells, and has a fast lysis speed, and at the same time, to overcome the disadvantages of low detection sensitivity and low accuracy of the detection rod in the prior art, to develop a fuel microbial detection rod specially adapted to be based on bioluminescence reaction, so that the fluorescence reaction is concentrated and stable,

the higher the content of ATP and the proportional relation between the ATP content and the Relative Luminescence Units (RLU), the higher the content of microorganisms, the higher the relative luminescence units; secondly, the mixture of apyrase, adenosine phosphate deaminase and copper sulfate can effectively remove free ATP in the sample; the octadecyl trimethyl ammonium chloride can effectively crack the microbial cells to release ATP in the microbial cells, the cracking speed is high, the cracking is complete, and the residual ATP hydrolase can be irreversibly inactivated; fourthly, the luciferase is freeze-dried powder and can be stored at normal temperature, so that the storage cost is saved; fifthly, the rod part of the detection rod adopts a thread design, so that the result is more stable; and sixthly, the detection tube contains a fluorescence aggregation reaction ball, so that fluorescence can be concentrated on the surface of the reaction ball, and the detection sensitivity, stability and accuracy are improved.

Disclosure of Invention

The invention makes up the defects of the traditional fuel microorganism detection method, provides the method for rapidly detecting the microorganisms in the fuel oil sample by the ATP bioluminescence method, eliminates the influence of free ATP in the sample, and improves the detection speed, sensitivity, precision and accuracy.

The invention aims to provide a detection method for rapidly detecting microorganisms in fuel oil by an ATP bioluminescence method and a special detection rod thereof.

Specifically, the method for detecting the microorganisms in the fuel oil by using the bioluminescence method comprises the following steps:

a) pulling out the swab from the detection rod, putting the swab into a sample to be detected, and stirring for 5 seconds;

b) the swab is inserted back into the detection rod again, and the handle is rotated downwards, so that the top of the swab sequentially penetrates through the upper layer of aluminum foil and the lower layer of aluminum foil of the first mixing chamber to enter the second mixing chamber of the detection tube, and the light is kept away;

c) holding the rod part of the detection rod, and shaking the detection rod for 3 times, wherein the detection tube contains a liquid reagent and a powder reagent; the first mixing chamber is filled with a liquid reagent which is an ATP hydrolytic agent and specifically consists of a mixture of apyrase, adenosine phosphate deaminase and copper sulfate, wherein the activities of the apyrase and the adenosine phosphate deaminase are respectively 0.035-0.045U/ml and 0.008-0.012U/ml, and the concentration of the copper sulfate is 0.4-0.8 g/L; the second mixing chamber is filled with a powder reagent which is a cell cracking agent, and the cell cracking agent is octadecyl trimethyl ammonium chloride, and the content of the octadecyl trimethyl ammonium chloride is 0.23-0.27 mg;

d) completely screwing out the swab, holding the rod part of the detection rod, and shaking the detection rod for 3 times to enable the reagent to enter a second mixing chamber of the detection tube; reacting through a fluorescence aggregation reaction ball in the detection tube;

e) the handle is screwed off again; this step is to prevent the swab from carrying away the reagent, keeping it entirely in the second mixing chamber;

f) the microtube cover is pulled out, the detection rod is immediately inserted into the photometer, and the whole detection tube is completely inserted into the sample chamber to read data.

Further, the rapid detection method of the invention also comprises the sample preparation steps:

a) to the extraction flask was added 20ml of sterile saline (0.8% NaCl) followed by 150ml of the fuel sample.

b) The lid was closed and mixed for at least 10 seconds. The extraction flask was inverted and held for a few seconds to allow the aqueous phase to settle to the bottom of the flask.

c) The stopper at the bottom of the extraction flask was removed and the aqueous phase was slowly added to the sterile sample flask. If microorganisms are present in the sample, the microorganisms will be distributed in the aqueous phase. It may be necessary to squeeze the extract collection bottle to drain all of the aqueous phase (25-30 ml).

Further, the preparation method of the liquid reagent comprises the following steps:

1) weighing 4.48g of trimethyl amino acid, 0.6g of copper sulfate, 0.146g of EDTA, 100mg of bovine serum albumin and 77mg of dithiothreitol, and adding the mixture into 600ml of water;

2) adding 4ml of 10U/ml apyrase and 1ml of 10U/ml adenosine phosphate deaminase, adjusting the pH to 7.8 with 10% sodium hydroxide, and adding distilled water to make the volume to 1 liter; the activity of the solution is respectively 0.04U/ml and 0.01U/ml;

3) the reagent is subpackaged into each detection, each tube is 1ml, and the pH value of the liquid reagent is 7.6-8.0.

Further, the preparation method of the powder reagent comprises the following steps: mu.g luciferase, 7.254ng D-luciferin sodium, 1.3146. mu.g magnesium sulfate and 0.25mg octadecyl trimethyl ammonium chloride were weighed and mixed well.

Further, the higher the Relative Luminescence Units (RLU) of the results interpretation samples, the more severe the contamination was

RLUM < 500,000RLU, indicating no contamination

RLUM is 500,000-1500,000, indicating suspected contamination

RLUM > 1,500,000RLU, indicating the presence of contamination.

The invention also aims to provide a detection rod for detecting microorganisms in fuel oil by a special bioluminescence method, which comprises the following components: the detection device comprises a swab main pipe 1, wherein a guide thread 2 is formed in the surface of the swab main pipe 1, a swab handle 10 is movably mounted on the surface of the swab main pipe 1, a detection pipe 6 is mounted at the bottom of the swab main pipe 1, a fluorescence aggregation reaction ball 61 is arranged at the bottom of an inner cavity of the detection pipe 6, the fluorescence aggregation reaction ball 61 is porous, and a microporous film 62 is wrapped on the surface of the fluorescence aggregation reaction ball 61; a first mixing chamber and a second mixing chamber are respectively arranged in the detection tube 6 from top to bottom and respectively contain a liquid reagent and a powder reagent.

The diameter of the fluorescence aggregation reaction ball is 3-5mm, the components are aluminum silicate, the aperture of the inner layer is larger and is 40-60nm, the surface layer is microporous, the aperture is uniform and is 8-15nm, and the pore volume is 0.06-0.10cm³Per g, the specific surface area is 50-65cm²/g。

Furthermore, 4 transparent ribs are uniformly arranged on the inner wall of the detection tube 6, the width of the detection tube is 0.2-0.2mm, and the length of the detection tube is 1.5-2.5 cm.

Further, the bottom fixed mounting that the swab was responsible for 1 has layer board 3, the bottom fixed mounting of layer board 3 has auxiliary pipe 4, the swab is responsible for 1 and auxiliary pipe 4 intercommunication, the bottom fixed mounting of auxiliary pipe 4 has installation cover 5, the bottom screw thread of installation cover 5 is installed and is detected pipe 6, the movable micro-tube cap 15 that cup joints in the detection pipe 6 outside.

Further, installation cover 5 and auxiliary 4 intercommunication, the leading truck 7 has all been welded to the both sides at layer board 3 top, constant head tank 8 has been seted up on the surface of swab handle 10, the inside movable mounting of leading truck 7 has the setting element 9 with constant head tank 8 looks adaptation, the inner wall fixed mounting of swab handle 10 has the power part 11 with 2 looks adaptations of direction screw thread, the long slide opening 12 with setting element 9 looks adaptations is seted up to one side of leading truck 7.

Further, a swab connecting rod 13 is fixedly installed at the top of the inner cavity of the swab handle 10, and the bottom end of the swab connecting rod 13 extends into the inner cavity of the secondary tube 4. The bottom end of the swab connecting rod 13 is fixedly provided with a sampling swab 14.

Further, the positioning element 9 includes a sliding block 91 sliding in the inner cavity of the long sliding hole 12 and a sliding groove 94 formed in the inner wall of the guide frame 7, ball sleeves 92 are welded on two sides of the sliding block 91, balls 93 are movably mounted in the inner cavity of the ball sleeves 92, and a spiral positioning pin 95 matched with the positioning groove 8 is connected to one side of the sliding block 91 through threads.

Further, one end, far away from the sliding block 91, of the spiral positioning pin 95 is fixedly connected with a twisting block 96, and the surface of the twisting block 96 is provided with anti-skid threads.

The invention has the beneficial effects that:

1. the invention discloses a detection rod special for detecting microorganisms in fuel oil by a bioluminescence method, which is based on bioluminescence reaction. The intensity of the light produced by the bioluminescent reaction is then measured using a luminometer, the intensity of the light being proportional to the amount of microbial ATP present in the fuel sample.

2. When the microorganism is detected, a user firstly unscrews the positioning piece, then rotates the swab handle, punctures the first sealing film and the second sealing film in sequence, and enters the first mixing chamber and the second mixing chamber in sequence, so that the detection ball with the detection substance is contacted and mixed with the detection substance, and the purpose of detecting the microorganism is achieved. The invention adopts the screw thread design, and the speeds of different operators rotating the swab are similar, so the reaction time of the sampling swab in the liquid is more consistent, and the stability is higher.

3. According to the invention, through the arrangement of the positioning piece, when the position of the swab handle moving downwards needs to be stopped at a proper position, a user can slide the sliding block up and down, the sliding block drives the ball sleeve to slide in the inner cavity of the sliding groove, meanwhile, the ball can roll along the inner wall of the sliding groove, and when the position is moved to a proper position corresponding to the positioning groove, the twisting block is screwed down, and the spiral positioning pin is inserted into the positioning groove, so that the purpose that the swab handle can be positioned at any position can be realized.

4. The invention discovers for the first time that the mixture of apyrase, adenosine phosphate deaminase and copper sulfate can effectively remove free ATP in a sample; the addition of copper sulfate can synergistically enhance the hydrolysis of apyrase and adenosine phosphate deaminase. Through continuous tests, the inventor finds that the relative luminescence unit is firstly reduced and then increased along with the increase of the concentration of copper sulfate, and when the addition amount is 0.6mg, the relative luminescence unit is the lowest, which indicates that the ATP hydrolytic agent has the strongest hydrolytic capability at the moment. Therefore, the present invention selects 0.6mg as the optimum amount of copper sulfate to be added as the free ATP hydrolysis agent.

5. The invention discovers for the first time that the octadecyl trimethyl ammonium chloride can effectively crack the microbial cells to release ATP in the microbial cells, the cracking speed is high, the cracking is complete, and the residual ATP hydrolase can be irreversibly inactivated.

6. The fluorescence aggregation reaction ball developed for the first time can concentrate fluorescence on the surface of the reaction ball, and the detection sensitivity, stability and accuracy are improved. Through different experimental comparisons, the diameter of the fluorescence aggregation reaction ball is 3-5mm, the inner diameter is 40-60nm, the surface layer is microporous, the aperture is uniform and is 8-15nm, and the pore volume is 0.06-0.10cm³Per g, the specific surface area is 50-65cm²The detection result is higher than that of the reaction ball without the addition of the fluorescence aggregation, which shows that the addition of the fluorescence aggregation reaction ball can amplify the fluorescence signalI.e. a higher sensitivity.

Drawings

FIG. 1 is a schematic view of the structure of a detection rod;

FIG. 2 is a top perspective view of the wand;

FIG. 3 is a bottom perspective view of the wand;

FIG. 4 is a perspective assembly view of a guide bracket of the detection rod;

FIG. 5 is an enlarged view of a portion of FIG. 4;

fig. 6 is a perspective assembly view of a sampling swab.

FIG. 7 is a schematic cross-sectional view of a fluorescence aggregation reaction ball in a detection tube.

FIG. 8 is a view showing a state of use of the detection bar;

FIG. 9 shows the results of detection by adding a fluorescent aggregation reaction ball

FIG. 10 detection results of fluorescence-free aggregation reaction beads

FIG. 11 relative luminescence unit RLU vs. ATP concentration

FIG. 12 correlation of microbial content with relative luminescence units

FIG. 13 shows the results of the detection by the method of the present invention

FIG. 14 test results of the conventional method

FIG. 15 shows the results of detection of positive samples by the method of the present invention

FIG. 16 detection results of positive samples in the conventional method

FIG. 17 shows the results of the detection by the method of the present invention

FIG. 18 shows the results of the conventional method

FIG. 19 SEM photograph of fluorescent aggregation reaction sphere surface

FIG. 20 Infrared Spectrum of fluorescent aggregation reaction spheres

FIG. 21 Infrared-NMR spectra of fluorescent aggregation reaction spheres

FIG. 22 Infrared nuclear magnetic resonance NMR spectra of fluorescent aggregation reaction spheres

Description of reference numerals: 1. a swab main tube; 2. a guide thread; 3. a support plate; 4. a secondary pipe; 5. installing a sleeve; 6. a detection tube; 61. a fluorescence aggregation reaction ball; 62. a microporous film; 7. a guide frame; 8. positioning a groove; 9. a positioning member; 10. a swab handle; 11. a power member; 12. a long slide hole; 13. a swab connecting rod; 14. sampling a swab; 15. a micro-tube cover; 91. a slider; 92. a ball sleeve; 93. a ball bearing; 94. a chute; 95. a screw positioning pin; 96. the block is twisted.

Detailed Description

Embodiments of the present invention are described below with reference to the drawings. Elements and features depicted in one drawing or one embodiment of the invention may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that the figures and description omit representation and description of components or processes that are not relevant to the present invention and that are known to those of ordinary skill in the art for the sake of clarity.

The invention is further described below with reference to the accompanying drawings.

Example 1

The embodiment of the application discloses a convenient detection stick swab handle to swab fastening release includes: swab is responsible for 1, and the swab is responsible for 1 surface and has seted up direction screw thread 2, and the swab is responsible for 1 movable surface mounting of 1 has swab handle 10, and the bottom fixed mounting that the swab is responsible for 1 has layer board 3, and the bottom fixed mounting of layer board 3 has auxiliary pipe 4, and the swab is responsible for 1 and auxiliary pipe 4 intercommunication, and the bottom fixed mounting of auxiliary pipe 4 has installation cover 5, and the bottom screw thread of installation cover 5 is installed and is detected pipe 6, the activity of detecting pipe 6 outside is cup jointed little tube cover 15, and the little tube cover plays the effect of light-resistant. A first mixing chamber and a second mixing chamber are respectively arranged in the detection tube 6 from top to bottom and respectively filled with a liquid reagent and a powder reagent; the bottom of the inner cavity of the detection tube 6 is provided with a fluorescence aggregation reaction ball 61, the fluorescence aggregation reaction ball 61 is porous, the surface of the fluorescence aggregation reaction ball 61 is wrapped by a layer of microporous film 62, the inner wall of the detection tube 6 is uniformly provided with transparent ribs, the microporous film 62 and the transparent ribs are arranged to increase the light gathering capacity, the detection precision of the detection rod can be improved, and the accuracy of detection data is guaranteed. The installing sleeve 5 is communicated with the auxiliary pipe 4, guide frames 7 are welded on two sides of the top of the supporting plate 3, positioning grooves 8 are formed in the surface of the swab handle 10, positioning pieces 9 matched with the positioning grooves 8 are movably installed inside the guide frames 7, power pieces 11 matched with the guiding threads 2 are fixedly installed on the inner wall of the swab handle 10, and long sliding holes 12 matched with the positioning pieces 9 are formed in one side of the guide frames 7. The top fixed mounting of swab handle 10 inner chamber has swab connecting rod 13, and the inner chamber of auxiliary pipe 4 is stretched into to the bottom of swab connecting rod 13, and the bottom fixed mounting of swab connecting rod 13 has sampling swab 14, and sampling swab 14's shape is spherical, and the one end fixedly connected with that sliding block 91 was kept away from to spiral locating pin 95 twists round piece 96, twists round piece 96's surface and is provided with anti-skidding line. The number of the positioning grooves 8 is not less than five, the positioning grooves 8 are distributed on the surface of the swab handle 10 in a step shape, the guide screw thread 2 is in a spiral shape, and the power piece 11 is in a spiral shape matched with the guide screw thread 2.

In actual use, when detecting microorganisms, a user firstly unscrews the positioning part 9, then rotates the swab handle 10, the swab handle 10 rotates in the inner cavity of the guide screw 2, the swab handle 10 slides downwards under the action of the guide screw 2 and the power part 11, and then drives the swab connecting rod 13 to move downwards, the swab connecting rod 13 drives the sampling swab 14 to move downwards, so that a sealing film at the top of the detection tube 6 is punctured and enters the first mixing chamber at the top of the inner cavity of the detection tube 6, then the positioning part 9 is screwed down, so that the spiral positioning pin 95 is inserted into the positioning groove 8, so as to position the swab handle 10, and limit the movement of the swab connecting rod 13 and the sampling swab 14, then the whole detection rod is held to shake, so that a detection substance is absorbed by the sampling swab 14, then the positioning part 9 is unscrewed, so that the spiral positioning pin 95 moves out of the inner cavity of the positioning groove 8, the locking is released, the swab handle 10 is rotated again, the swab connecting rod 13 drives the sampling swab 14 to puncture the two sealing films again, and the two sealing films enter the second mixing chamber at the bottom of the inner cavity of the detection tube 6, so that the sampling swab 14 with the detection substance is contacted and mixed with the detection substance, and the purpose of detecting microorganisms is achieved.

Example 2

With reference to fig. 1 and 5, the positioning element 9 includes a sliding block 91 sliding in the inner cavity of the long sliding hole 12 and a sliding slot 94 formed in the inner wall of the guiding frame 7, ball sleeves 92 are welded on both sides of the sliding block 91, balls 93 are movably mounted in the inner cavity of the ball sleeves 92, one side of the sliding block 91 is connected with a spiral positioning pin 95 adapted to the positioning slot 8 through threads, the positioning element 9 is arranged, when the swab handle 10 moves down, the position needs to be stopped at a proper position, the sliding block 91 can slide up and down by a user, the sliding block 91 drives the ball sleeves 92 to slide in the inner cavity of the sliding slot 94, meanwhile, the balls 93 roll against the inner wall of the sliding slot 94, move to a proper position corresponding to the positioning slot 8, the twisting block 96 is screwed down, the spiral positioning pin 95 is inserted into the positioning slot 8, so that the swab handle 10 can be positioned at any position.

Example 3

Preparation of liquid reagent in detection tube:

1) 4.48g of trimethylamino acid, 0.6g of copper sulfate, 0.146g of EDTA, 100mg of bovine serum albumin, and 77mg of dithiothreitol were weighed and added to 600ml of water.

2) 4ml of 10U/ml apyrase and 1ml of 10U/ml adenosine phosphate deaminase were added, the pH was adjusted to 7.8 with 10% sodium hydroxide and made up to 1 liter with distilled water. The activity was adjusted to 0.04U/ml and 0.01U/ml, respectively.

3) This was dispensed into each assay, 1ml per tube.

Preparation of powder reagent in detection tube:

1) mu.g luciferase, 7.254ng D-luciferin sodium, 1.3146. mu.g magnesium sulfate and 0.25mg octadecyl trimethyl ammonium chloride were weighed and mixed well.

Preparation of a detection tube:

1) the powdered reagent was added to a 2ml screw plastic test tube and 1ml of the liquid reagent was sealed in aluminum foil on the top of the test tube.

Assembling the detection rod:

1) the test tube, threaded stem and swab are assembled.

Experimental example 1

This experimental example consists in studying the optimal activity of apyrase and adenosine phosphate deaminase in ATP hydrolysing agents.

Dissolving ATP disodium salt with Tris-EDTA buffer solution to obtain ATP concentration of 10^-7And M. 1ml of the prepared ATP solution was taken, apyrase (A) and adenosine phosphate deaminase (B) were added thereto, and reacted for 30 seconds,then, a fluorescent reagent (1. mu.g luciferase, 7.254ng D-luciferin sodium, and 1.3146. mu.g magnesium sulfate) was added, Relative Luminescence Units (RLU) were measured using a fluorometer, 5 replicates of each assay were taken, and the results were averaged and are shown in Table 1. According to the law that the content of microorganisms in fuel is proportional to the content of ATP, the content of ATP is also proportional to the Relative Luminescence Units (RLU), so that the higher the content of microorganisms, the higher the relative luminescence units.

TABLE 1 results of detection of different enzyme activities

As can be seen from Table 1, when the activity of apyrase is 0.04-0.06U/L and the activity of adenosine phosphate deaminase is 0.01-0.02U/L, the measured fluorescence intensity is very low, indicating that the enzyme has very strong hydrolytic ability and no significant difference in fluorescence intensity, indicating that the hydrolytic ability is similar, therefore, the mixture of apyrase with minimum activity of 0.04U/L and adenosine phosphate deaminase of 0.01U/L is selected as the main component of the free ATP hydrolytic agent.

Experimental example 2

The experimental example consists in studying the concentration of copper sulphate.

Dissolving ATP disodium salt with Tris-EDTA buffer solution to obtain ATP concentration of 10^-7And M. 1ml of the prepared ATP solution was taken, copper sulfate was added in amounts of 0mg, 0.2mg, 0.4mg, 0.6mg, 0.8mg and 1.0mg, respectively, then 0.04U/L apyrase and 0.01U/L adenosine phosphate deaminase were added, reaction was carried out for 30 seconds, then fluorogenic reagents (1. mu.g luciferase, 7.254ng D-luciferin sodium and 1.3146. mu.g magnesium sulfate) were added, Relative Luminescence Units (RLU) were measured with a fluorometer, 5 replicates of each assay were made, and the results were averaged as shown in Table 2.

TABLE 2 results of measurements of different copper sulfate concentrations

	0	0.2	0.4	0.6	0.8	1.0
							1	13309	10065	8909	6348	7990	9017
2	12985	10101	8187	6501	7765	8867
							3	13199	11684	8658	6587	8102	8905
4	12768	11548	8800	6618	8017	8991
							5	13078	10930	8759	6499	8211	9235
Mean value of	13067.8	10865.6	8662.6	6510.6	8017	9003

As can be seen from Table 2, the relative luminescence units decreased and then increased with the increase in the copper sulfate concentration, and when the addition amount was 0.6mg, the relative luminescence units were the lowest, indicating that the hydrolysis ability of the ATP hydrolyzing agent was the strongest at this time. Therefore, 0.6mg was selected as the optimum amount of copper sulfate to be added as a free ATP hydrolyzing agent.

Experimental example 3

The experimental example was conducted to investigate the kind and content of the optimal cell lysis agent.

5 kinds of fuel characteristic microorganisms, namely, resin ascomycetes (Cladosporum Resinae), Aspergillus Niger (Aspergillus Niger), Chaetomium Globosum (Chaetomium Globosum), pullulan producing bacteria (Aerobasidium Pullulans) and Fusarium Moniliforme (Fusarium Moniliforme) are mixed in equal proportion to form mixed bacteria liquid. And adding 1ml of liquid reagent into 80 mu l of mixed bacterial liquid, then respectively adding a certain amount of octadecyl trimethyl ammonium chloride (STAC), dodecyl dimethyl Benzyl Ammonium Bromide (BAB) and benzalkonium chloride (BAC), oscillating for 3 seconds, then adding 1 mu g of luciferase, 7.254ng of D-fluorescein sodium and 1.3146 mu g of magnesium sulfate, rapidly oscillating for 2 seconds, detecting the RLU value by using a fluorescence detector, making 5 parallels for each group of experiments, and averaging the results. The results are shown in Table 3.

TABLE 3 influence of different cracking agents and addition amounts on the test results

Addition amount (mg)	0.10	0.15	0.20	0.25	0.30	0.35	0.40
								STAC(RLU)	11837	315386	506528	658972	613175	524268	308912
BAB(RLU)	8081	182561	367967	501523	483312	356097	328769
								TCA(RLU)	7636	160637	358793	460928	510347	413205	220993

As can be seen from table 3, the relative luminescence units obtained by using octadecyl trimethyl ammonium chloride (STAC) as the cracking agent are generally higher than those obtained by using benzalkonium bromide (BAB) and benzalkonium chloride (BAC) as the cracking agent, indicating that octadecyl trimethyl ammonium chloride (STAC) releases more ATP as the cracking agent and thus the cracking effect is better, and when the addition amount of octadecyl trimethyl ammonium chloride (STAC) is 0.25mg, the obtained relative luminescence units are the highest, indicating that the cracking effect is the best.

Experimental example 4

The experimental example consists in studying the optimum pH of a liquid reagent.

The pH of the liquid reagents was adjusted to 6.0, 7.0, 7.8, 8.5, 9.5 with 10% HCl or 10% NaOH. 80 μ l of the mixed bacterial solution prepared in experimental example 2 was taken, 1ml of liquid reagent was added, 0.25mg of octadecyl trimethyl ammonium chloride was added, after shaking for 3 seconds, 1 μ g of luciferase, 7.254ng of D-luciferin sodium and 1.3146 μ g of magnesium sulfate were added, shaking was rapidly performed for 2 seconds, the RLU value was detected by a fluorescence detector, 3 replicates of each experiment were performed, and the results were averaged. The results are shown in Table 4.

TABLE 4 influence of pH on the assay results

pH	6.0	7.0	7.8	8.5	9.5
						RLU	518092	601287	658972	623201	456832

As can be seen from Table 4, the relative luminescence unit was highest at pH 7.8, indicating that the cleavage effect of the cleavage agent was the best and the inhibition of the fluorescence reaction was the least at this pH, and therefore, pH 7.8 was selected as the optimum pH.

Experimental example 5

This experimental example consists in studying the influence of the design of the helix of the stem on the stability of the results.

Selecting a fuel sample, adding fuel polluted by microorganisms into the fuel sample according to the proportion of 0.2%, 1.0%, 3.0% and 7.0%, respectively using the detection rod and the unthreaded detection rod, extracting and detecting the sample according to the method of the invention, and performing 5 parallels on each sample, wherein the detection results are shown in tables 5-6.

TABLE 5 coefficient of variation of the results of threaded test bars

TABLE 6 coefficient of variation of the results of the threadless test bars

As can be seen from table 5 and table 6, the coefficient of variation obtained by using the threaded test rod of the present invention is 2.89 to 5.26%, and the coefficient of variation of the unthreaded test rod is 12.19 to 26.71%, so that the coefficient of variation obtained by using the threaded test rod is smaller, indicating that the stability of the result is higher. With the screw thread design, the speed of rotation of the swab by different operators is similar, so that the reaction time of the sampling swab in the liquid is more consistent, while the non-screw thread design results in different speeds of insertion of the swab by different operators and thus higher stability.

Experimental example 6

The experimental example is to study the influence of the fluorescence aggregation reaction ball on the detection result.

The effect on sensitivity.

ATP-free water was used to prepare ATP disodium salt stock solution. Prepared with stock solution to a concentration of 10^-17M is ATP solution. 1.0mL of each concentration of ATP (sodium salt) solution was added to a sterile, ATP-free, 1.5mL small centrifuge tube. Measurements were performed and read 5 times for each method according to the detection method of the present invention and the method without the addition of the fluorescent aggregation reaction beads, respectively. The results are shown in Table 7.

TABLE 7 influence of fluorescent aggregation reaction spheres on sensitivity

As can be seen from Table 7, the detection result of the fluorescence aggregation reaction ball is higher than that of the fluorescence aggregation reaction ball, which indicates that the fluorescence signal can be amplified by adding the fluorescence aggregation reaction ball, i.e., the sensitivity is higher.

Influence on stability of results

ATP-free water was used to prepare ATP disodium salt stock solution. The stock solution was used to prepare the following ATP solution 10^-11M,10^-13M，10^-15M，10^-17And M. 1.0mL of each ATP (sodium salt) solution was added to a sterile, ATP-free, 1.5mL small centrifuge tube. The assay and reading were performed according to the assay method of the present invention and the method without the addition of the fluorescent aggregation beads, respectively, and each concentration was repeated 5 times. The results are shown in Table 8.

TABLE 8 influence of fluorescent aggregation reaction spheres on stability

As shown in Table 8, for ATP solutions with different concentrations, the coefficient of variation of the RLU values obtained by the detection method is 3.83-4.85%, and the coefficient of variation of the RLU values obtained by the detection method adopting the reaction-free spheres is 8.48-16.71%, so that the coefficient of variation of the RLU values obtained by the detection method adopting the reaction-free spheres is lower than that of the RLU values obtained by the detection method adopting the reaction-free spheres, and the method disclosed by the invention is higher in stability.

Influence on the accuracy of the results

To 10 fuel samples were added ascosphates resinifera (Cladosporum Resinae), 2 samples were negative, 7 samples were weakly positive and 1 sample was positive by MicrotestP detection. The assay and reading were performed using the assay method of the invention and the method without the addition of the fluorescent aggregation reaction beads, respectively, and each sample was repeated 10 times. The results are shown in FIGS. 9-10.

As can be seen from FIG. 9, the detection results of the negative samples are all lower than 500000, the detection results of the weak positive samples are all 500000-1500000, and the detection results of the positive samples are all higher than 1500000, so the detection results are consistent with the MicrotestP method.

As can be seen from fig. 10, when the method without adding the fluorescent aggregation reaction beads is used for detection, 6 detection results in the weakly positive sample are less than 50000 (negative), and 4 detection results in the positive sample are less than 1500000 (suspected positive), so that the inhibition rate of the detection result with the MicrotestP method is 90%.

Therefore, the accuracy of the result can be obviously improved by adding the fluorescence aggregation reaction ball.

Experimental example 7

This example is intended to investigate the relationship between Relative Luminescence Units (RLU) and ATP concentration.

ATP-free water was used to prepare ATP disodium salt stock solution. The stock solution was used to prepare the following ATP solution 10^-11M，6×10^- ¹²M,3×10^-12M,3×10^-13M, 0M. 1.0mL of each ATP (sodium salt) solution was added to a sterile, ATP-free, 1.5mL small centrifuge tube. It is recommended that each concentration be repeated at least 5 times. Measured and read according to the detection method of the present invention. And (3) drawing a standard curve by taking the ATP concentration as an abscissa and the detection result as an ordinate, wherein the standard curve is shown in a figure 10.

As can be seen from fig. 11, the Relative Luminescence Unit (RLU) becomes higher and higher as the ATP concentration increases, and the ATP concentration has a linear relationship with the Relative Luminescence Unit (RLU).

Experimental example 8

This example consists in studying the relationship between the microbial content and the Relative Luminescence Units (RLU).

Selecting a fuel sample, adding the fuel polluted by microorganisms into the fuel sample according to the proportion of 0.2 percent, 1.0 percent, 3.0 percent and 7.0 percent, and extracting and detecting the sample according to the method of the invention, wherein the detection result is shown in figure 12. The results show that the microorganism content and the Relative Luminescence Unit (RLU) have linear relation and good correlation, and R is²Is 0.9831.

Experimental example 9

This experimental example is intended to investigate the accuracy of the method of the invention.

And (3) negative sample verification: fuel samples that were negative to MicrotestP were tested using the method of the invention and the general method and the results are shown in FIGS. 13-14.

The common method comprises the following steps: remove the small pipette from the fuel detection kit. The capture solution was transferred from the reservoir of the fuel detection stick to the sample vial containing the sample using a sterile small pipette. The interior of the pipette is flushed with the sample, ensuring maximum transfer of the capture solution to the sample. The sample vial was capped and shaken vigorously for 30 seconds. The sample bottle was placed on a horizontal surface and allowed to stand for 5 minutes. The photometer is powered on and self-testing has been successfully completed and analysis can be performed.

As can be seen in FIGS. 13-14, 95% of negative samples were < 1200K RLU when tested using the methods described herein; 95% of negative samples were < 320RLU, as determined by the usual methods. Thus, the sensitivity of the method of the invention is higher.

And (3) positive sample verification: the fuel samples were separately added with ascosphaera resinifera (Cladosporum Resinae), Aspergillus Niger (Aspergillus Niger), Chaetomium Globosum (Chaetomium Globosum), Protaminobacter Pullulans (Aerobasidium Pullulans) and Fusarium Moniliforme (Fusarium Moniliforme), and the results were weakly positive by MicrotestP method. The results of the tests were shown in FIGS. 15-16, which were performed by 5 operators using the method of the present invention and the conventional method.

As can be seen from FIGS. 15-16, for weakly positive samples, 40% of the test results obtained by the method of the present invention are positive, and 25% of the test results obtained by the conventional method are positive; for positive samples, the detection results of the method are positive, most of the detection results of the common method are positive, and the small part of the detection results are negative. Therefore, the accuracy of the method is better than that of the conventional method.

As can be seen from FIGS. 17-18, for the methods of the present invention, the negative samples all tested less than 1200K RLU, and the positive samples all tested more than 1500K RLU; for the conventional method, the detection results of the negative samples are all lower than 450RLU, the detection results of about 80% of the positive samples are greater than 400RLU, and the detection results of about 20% of the positive samples are less than 250RLU, so that the method has better identification capability.

Experimental example 10

This experimental example is intended to study the precision of the present invention.

A, B, C, D four kinds of fuel (A: middle aviation kerosene, B: middle petrochemical 97 # vehicle gasoline (II), C: middle petrochemical-50 # vehicle diesel, D: middle petrochemical biodiesel) are selected and respectively diluted with bacterial liquid with the dilution multiple of 1 × 10^-4、6×10^-5、3×10^-5、1×10^-5、6×10^-6、3×10^-6、1×10^-6、6×10^-7The total number of oil samples used for the reproducibility test was 37. Performing the test according to the method of the invention under a repetitive condition, and expressing the measurement result by RLU; the test results are shown in Table 9.

TABLE 9 results of the repeatability tests

In RLU

As can be seen from Table 9, the differences for all samples of the method of the invention do not exceed the reproducibility limit (r) required for the standard method, which is seen to have a better reproducibility.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, devices, means, methods, or steps.

Claims

1. A method for detecting microorganisms in fuel oil by a bioluminescence method is characterized by comprising the following steps:

b) the swab is inserted back into the detection rod again, and the handle is rotated downwards, so that the top of the swab sequentially penetrates through the two layers of aluminum foils above and below the first mixing chamber and enters the second mixing chamber of the detection tube, and the light is kept away;

c) holding the rod part of the detection rod, and shaking the detection rod for 3 times, wherein the detection tube contains a liquid reagent and a powder reagent; the first mixing chamber is filled with a liquid reagent which is an ATP hydrolytic agent and specifically consists of a mixture of apyrase, adenosine phosphate deaminase, copper sulfate, trimethyl amino acid, EDTA, bovine serum albumin and dithiothreitol, wherein the activities of the apyrase and the adenosine phosphate deaminase are respectively 0.035-0.045U/ml and 0.008-0.012U/ml, and the concentration of the copper sulfate is 0.4-0.8 g/L; the second mixing chamber is filled with a powder reagent which is luciferase, D-luciferin sodium, magnesium sulfate and a cell cracking agent, and the cell cracking agent is octadecyl trimethyl ammonium chloride with the content of 0.23-0.27 mg;

d) completely screwing out the swab, holding the rod part of the detection rod, shaking the detection rod for 3 times, and allowing the fluorescence aggregation reaction ball in the detection tube to react, wherein the surface of the fluorescence aggregation reaction ball is coated with a layer of microporous film; the diameter of the fluorescence aggregation reaction ball is 3-5mm, the components are aluminum silicate, the aperture of the inner layer is larger and is 40-60nm, the surface layer is microporous, and the aperture is uniform and is 8-15 nm;

e) the handle is screwed off again;

2. The method of claim 1, wherein the liquid reagent is prepared by the following method:

1) weighing 4-5g of trimethyl amino acid, 0.5-0.7g of copper sulfate, 0.1-0.3 g of EDTA, 95-105mg of bovine serum albumin and 72-82mg of dithiothreitol, and adding into 600-700ml of water;

2) adding 4ml of 10U/ml apyrase and 1ml of 10U/ml adenosine phosphate deaminase, adjusting the pH to 7.5-8.0 with 10% sodium hydroxide, and adding distilled water to make the volume to 1 liter; the activity of the solution is respectively 0.04U/ml and 0.01U/ml;

3) subpackaging into each detection tube, wherein each tube is 1ml, sealing in an aluminum foil layer of the detection tube, and the pH of the liquid reagent is 7.6-8.0;

alternatively, the preparation method of the powder reagent is as follows: mu.g luciferase, 7.254ng D-luciferin sodium, 1.3146. mu.g magnesium sulfate and 0.25mg octadecyl trimethyl ammonium chloride were weighed and mixed well.

3. The method of claim 1 or 2, wherein the detection bar comprises: swab is responsible for (1), direction screw thread (2) have been seted up on the surface that swab is responsible for (1), the surface movable mounting that swab is responsible for (1) has swab handle (10), detection tube (6) are installed to the bottom that swab is responsible for (1), the bottom of detection tube (6) inner chamber is provided with fluorescence gathering reaction ball (61), and fluorescence gathering reaction ball (61) are poroid, set up first mixing chamber and second mixing chamber respectively from top to bottom in detection tube (6).

4. A special detection rod for detecting microorganisms in fuel oil by a bioluminescence method is characterized by comprising: the detection device comprises a swab main pipe (1), wherein a guide thread (2) is formed in the surface of the swab main pipe (1), a swab handle (10) is movably mounted on the surface of the swab main pipe (1), a detection pipe (6) is mounted at the bottom of the swab main pipe (1), a fluorescence aggregation reaction ball (61) is arranged at the bottom of an inner cavity of the detection pipe (6), the fluorescence aggregation reaction ball (61) is porous, and a layer of microporous film (62) is wrapped on the surface of the fluorescence aggregation reaction ball (61); the diameter of the fluorescence aggregation reaction ball (61) is 3-5mm, the components are aluminum silicate, the aperture of the inner layer is larger and is 40-60nm, the surface layer is microporous, the aperture is uniform and is 8-15nm, and a first mixing chamber and a second mixing chamber are respectively arranged in the detection tube (6) from top to bottom.

5. The detection rod according to claim 4, wherein the fluorescence aggregation reaction ball (61) has a pore volume of 0.06-0.10cm³Per g, the specific surface area is 50-65cm²/g。

6. The detection rod according to claim 4, characterized in that the inner wall of the detection tube (6) is uniformly provided with 4 transparent ribs, the width of the transparent ribs is 0.1-0.2mm, and the length of the transparent ribs is 1.5-2.5 cm.

7. The detection rod according to claim 4, characterized in that a support plate (3) is fixedly installed at the bottom of the swab main pipe (1), a secondary pipe (4) is fixedly installed at the bottom of the support plate (3), the swab main pipe (1) is communicated with the secondary pipe (4), an installation sleeve (5) is fixedly installed at the bottom of the secondary pipe (4), a detection pipe (6) is installed at the bottom of the installation sleeve (5) in a threaded manner, and a micro-pipe cover (15) is movably sleeved outside the detection pipe (6).

8. The detection rod according to claim 7, wherein the mounting sleeve (5) is communicated with the auxiliary pipe (4), guide frames (7) are welded on both sides of the top of the support plate (3), a positioning groove (8) is formed in the surface of the swab handle (10), a positioning piece (9) matched with the positioning groove (8) is movably mounted inside the guide frame (7), a power piece (11) matched with the guide thread (2) is fixedly mounted on the inner wall of the swab handle (10), and a long sliding hole (12) matched with the positioning piece (9) is formed in one side of the guide frame (7); the swab handle is characterized in that a swab connecting rod (13) is fixedly mounted at the top of the inner cavity of the swab handle (10), the bottom end of the swab connecting rod (13) extends into the inner cavity of the auxiliary pipe (4), and a sampling swab (14) is fixedly mounted at the bottom end of the swab connecting rod (13).

9. The detection rod according to claim 8, wherein the positioning element (9) comprises a sliding block (91) sliding in the inner cavity of the long sliding hole (12) and a sliding groove (94) formed in the inner wall of the guide frame (7), ball sleeves (92) are welded on two sides of the sliding block (91), balls (93) are movably mounted in the inner cavity of the ball sleeves (92), and a spiral positioning pin (95) matched with the positioning groove (8) is in threaded connection with one side of the sliding block (91).

10. The detection rod according to claim 9, wherein a twisting block (96) is fixedly connected to one end of the spiral positioning pin (95) far away from the sliding block (91), and the surface of the twisting block (96) is provided with anti-skid grains.