阅读说明：本技术 一种快速初步筛查污染奶样中支原体的系统及其方法 (System and method for rapidly and primarily screening mycoplasma in polluted milk sample ) 是由 潘子豪 王凯民 俞菊仙 于 2021-07-19 设计创作，主要内容包括：本发明涉及一种快速初步筛查污染奶样中支原体的系统及其方法,包括离心机、过滤器、培养皿、色度检测仪,其筛查方法包括奶液预处理,培养检测。本发明利用液体培养基对预处理后的牛乳样本进行培养通过显色反应指示实验结果,较传统的检测方法极大的提高了检测效率,降低了检测成本,达到快速筛查致病病原及对牛传染性乳房炎进行初步诊断的目的,有效减少牧场或者乳企中乳汁的初步筛查的工作成本及劳动强度,并可及时发现可疑的奶样,从而提高了鲜奶质量检测的精度和效率。(The invention relates to a system and a method for rapidly and primarily screening mycoplasma in a polluted milk sample, wherein the system comprises a centrifugal machine, a filter, a culture dish and a chromaticity detector, and the screening method comprises milk liquid pretreatment and culture detection. According to the invention, the liquid culture medium is used for culturing the pretreated milk sample, and the experimental result is indicated through the chromogenic reaction, so that compared with the traditional detection method, the detection efficiency is greatly improved, the detection cost is reduced, the purposes of rapidly screening pathogenic pathogens and preliminarily diagnosing infectious bovine mastitis are achieved, the working cost and labor intensity of preliminary screening of milk in a pasture or a breast enterprise are effectively reduced, suspicious milk samples can be found in time, and the precision and efficiency of fresh milk quality detection are improved.)

1. A system for rapidly and primarily screening mycoplasma in a polluted milk sample is characterized in that: the system for rapidly and preliminarily screening mycoplasma in a polluted milk sample comprises a centrifuge (1), a filter (2), culture dishes (3) and a chromaticity detector (4), wherein the centrifuge (1) is connected with at least one filter (2) through a guide pipe, the filter (2) is connected with a plurality of culture dishes (3) in addition, the culture dishes (3) are connected in parallel, the chromaticity detector (4) is located above the culture dishes (3), and the optical axis of the chromaticity detector and the upper end of the culture dishes (3) form an included angle of 30-90 degrees.

2. The system for rapid preliminary screening of contaminated milk samples for mycoplasma according to claim 1, wherein: the culture dish (3) comprises a bearing frame (31), a bearing tray (32), a culture tray (33), a transparent sealing cover (34), a temperature sensor (35), a one-way valve (36), an ultrasonic oscillation mechanism (37), a silica gel heating bar (38) and a control circuit (39), wherein the bearing frame (31) is a columnar frame structure with the axis vertical to the horizontal plane, the bearing tray (32) is a closed annular structure with the cross section shaped like a U, is embedded in the upper end surface of the bearing frame (31) and is coaxially distributed with the bearing frame (31), the culture tray (33) is embedded in the upper end surface of the bearing tray (32) and is abutted against the bottom of the bearing tray (32) and is coaxially distributed, the culture tray (33) and the transparent sealing cover (34) are both of U-shaped groove-shaped structures with the cross section, and the transparent sealing cover (34) covers the upper end surface of the culture tray (33) and forms a closed cavity structure with the culture tray (33), the side surface of the transparent sealing cover (34) is hinged with the upper end surface of the bearing frame (31) through a turntable mechanism (5), a liquid filling port (6) and an exhaust port (7) are arranged on the transparent sealing cover (34), the liquid filling port (6) and the exhaust port (7) are symmetrically distributed on two sides of the axis of the culture disc (33) and form an included angle of 0-60 degrees with the axis of the culture disc (33), the liquid filling port (6) and the exhaust port (7) are both connected with a one-way valve (36), the temperature sensor (35) is embedded in the bearing tray (32), is coaxially distributed with the bearing tray (32) and is connected with the inner side surface of the bearing tray (32) through an elastic connecting mechanism (8), at least one silica gel heating strip (38) is embedded in the upper surface of the groove bottom of the bearing tray (32) and is a closed annular structure coaxially distributed with the bearing tray (32), and the ultrasonic oscillation mechanism (37) is embedded in the outer surface of the side wall of the bearing tray (32), and the control circuits (39) are embedded on the outer surface of the bearing rack and are respectively and electrically connected with the temperature sensor (35), the one-way valve (36), the ultrasonic oscillation mechanism (37), the silica gel heating strip (38) and the turntable mechanism (5).

3. A method for screening mycoplasma in polluted milk samples of a system for rapidly and primarily screening mycoplasma in polluted milk samples is characterized by comprising the following steps: the screening method for rapidly and primarily screening mycoplasma in polluted milk samples comprises the following steps:

s1, pretreating milk, taking 1-5 ml as a milk sample, centrifuging at least one milk sample by a centrifuge (1), filtering the centrifuged milk sample by a filter (2) at 0.40-0.45 mu m, and collecting filtrate for later use;

and S2, culturing and detecting, namely adding the filtrate collected in the step S1 and a liquid culture medium into a culture dish (3) for culturing, and detecting by a colorimetric method through a colorimetric detector (4) after culturing for 20-60 minutes to obtain a mycoplasma detection result.

4. The screening method for rapid preliminary screening of mycoplasma in contaminated milk samples according to claim 3, wherein the screening method comprises the following steps: the liquid culture medium comprises the following components in parts by weight: 10% -15% of PPLO broth, 5% -11% of compound horse serum, 13% -21% of 25% yeast extract, 2.3% -4.5% of ampicillin, 2.1% -3.5% of vancomycin, 3% -6% of glucose, 0.5% -1.8% of sodium pyruvate, 1.5% -3.1% of bovine lung digest, 0.3% -2.3% of 1% of phenol red, 0-11% of compound Erie's medium and the balance of ultrapure water.

5. The screening method for rapid preliminary screening of mycoplasma in contaminated milk samples according to claim 4, wherein the screening method comprises the following steps: the ampicillin is 5-8 ten thousand units.

6. The screening method for rapid preliminary screening of mycoplasma in contaminated milk samples according to claim 4, wherein the screening method comprises the following steps: the preparation method of the 25% yeast extract comprises the following steps:

dissolving fresh yeast in ultrapure water with the mass 1.5-5 times that of the fresh yeast, uniformly stirring, fully dissolving the fresh yeast, boiling for 15min, quickly cooling to the normal temperature at the speed of 10-20 ℃/min, centrifugally separating cooling liquid at the speed of 3500-5000 r/min for 15min, extracting supernatant, boiling the supernatant again for 15min, quickly cooling to the normal temperature at the speed of 10-20 ℃/min, filtering by using a K-type clarifying filter, filtering by using a 0.22-micron sterile filter membrane for sterilization, taking filtrate, and storing the filtrate at the constant temperature of 4 ℃.

7. The screening method for rapid preliminary screening of mycoplasma in contaminated milk samples according to claim 4, wherein the screening method comprises the following steps: the preparation method of the cattle lung digestive juice comprises the following steps:

firstly, boiling cattle lungs, crushing healthy cattle lungs, enabling the particle size of cattle lung particles to be not more than 1 mm, mixing the cattle lung particles with ultrapure water with the mass 2.5-6 times of that of the cattle lung particles, heating the mixed solution on an electric furnace to 60-80 ℃, and continuously stirring;

secondly, preparing a mixture, namely heating and preserving heat for 1-10 minutes, adding sodium carbonate accounting for 0.3-1.1% of the total amount of the mixed solution into the mixed solution, continuously preserving heat and stirring for at least 1 minute, then cooling to 45 ℃, adding a pancreatin solution accounting for 0.8-2.1% of the total amount of the mixed solution into the mixed solution again, finally carrying out water bath on the mixed solution for 3 hours at the constant temperature of 45 ℃, removing grease on the liquid level in the water bath process, and simultaneously adding concentrated hydrochloric acid accounting for 0.9-1.8% of the total amount of the mixed solution into the mixed solution and uniformly stirring;

and thirdly, filtering and collecting, boiling the mixed liquor prepared in the second step for 15min, cooling to normal temperature, filtering by double-layer gauze, centrifugally separating the filtrate for 15min at the speed of 2500-4000 r/min, collecting the filtrate, filtering the filtrate by using filter paper, boiling the filtrate filtered by the filter paper for 15min, cooling to room temperature, adjusting the pH value of the mixed liquor to 7.8 by using NaOH solution, sterilizing at 121 ℃ for 15min, and storing at constant temperature of 4 ℃.

8. The screening method for fast and preliminarily screening mycoplasma in polluted milk samples according to claim 3 or 4, wherein the screening method comprises the following steps: the preparation method of the liquid culture medium comprises the following steps:

the first step, the preparation treatment, namely, fully mixing PPLO broth, sodium pyruvate, MEM, bovine lung digestive juice, 1% phenol red and ultrapure water, adjusting the pH value to 7.8, then sterilizing at 121 ℃ for 15min, and cooling to 50-55 ℃;

and secondly, performing secondary treatment, namely adding the compound horse serum, the yeast leaching solution, ampicillin and vancomycin into the mixed solution prepared in the first step, uniformly mixing, and then regulating the pH value of the mixed solution to 7.8 to obtain a finished product.

Technical Field

The invention relates to a milk mycoplasma detection method, and belongs to the technical field of biological culture medium preparation and biological detection.

Background

Infectious mastitis: is the clinical or recessive mastitis of the dairy cow caused by the infection of infectious pathogenic bacteria, and is characterized in that the clinical infection rate gradually rises after the infectious mastitis infects animals; the pathogen can be infected by apparatus, personnel, even feed and water source. After the mycoplasma bovis infects the first-born cows, the later-period multiparous lactation yield of the infected animals is reduced, and high-yield cows are changed into low-yield cows. Currently, staphylococcus aureus and streptococcus agalactiae are all pathogenic pathogens of infectious mastitis. In addition, mycoplasma bovis (m. bovis) is also a common pathogen of bovine infectious mastitis, and mycoplasma bovis can cause local inflammation of cattle, such as inflammation of lung, breast, reproductive tract, joint and even sterility. It is mainly responsible for mastitis, resulting in a sharp reduction in lactation.

At present, common mycoplasma bovis screening and diagnosing methods for infectious bovine mastitis mainly comprise a colony observation method, a nucleic acid detection method, a probe method, a fluorescence color development method and a color development method, and although the requirements of detection operation can be met, the detection efficiency is low, the detection operation precision is poor, and the cost and labor intensity during the detection operation are relatively high.

Therefore, in order to solve the problem, a system and a method for rapidly and primarily screening mycoplasma in polluted milk samples are urgently needed to be developed so as to meet the requirement of practical use.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a system and a method for rapidly and primarily screening mycoplasma in a polluted milk sample.

The utility model provides a system for quick preliminary screening pollutes mycoplasma in milk appearance, includes centrifuge, filter, culture dish, colourity detector, wherein centrifuge passes through the honeycomb duct and is connected with at least one filter, the filter is connected with a plurality of culture dishes in addition, and each culture dish is parallelly connected each other, colourity detector is located the culture dish top, and its optical axis is 30-90 contained angles with culture dish upper end face.

Preferably, the culture dish comprises a bearing frame, a bearing tray, a culture disc, a transparent sealing cover, a temperature sensor, a one-way valve, an ultrasonic oscillation mechanism, a silica gel heating bar and a control circuit, wherein the bearing frame is a columnar frame structure with an axis vertical to the horizontal plane, the bearing tray is a closed annular structure with a U-shaped cross section, is embedded in the upper end surface of the bearing frame and is coaxially distributed with the bearing frame, the culture disc is embedded in the upper end surface of the bearing tray and is abutted against and coaxially distributed with the bottom of the bearing tray, the culture disc and the transparent sealing cover are both of a U-shaped groove-shaped cross section, the transparent sealing cover is coated on the upper end surface of the culture disc and forms a closed cavity structure with the culture disc, the side surface of the transparent sealing cover is hinged with the upper end surface of the bearing frame through a rotary table mechanism, and the transparent sealing cover is provided with a liquid filling opening and an exhaust opening, and filling opening and gas vent symmetric distribution are in cultivateing a set axis both sides to be 0-60 contained angles with cultivateing a set axis, and filling opening and gas vent all are connected with a check valve, temperature sensor inlays in bearing the tray, with bearing the tray coaxial distribution and be connected through elastic connection mechanism and bearing the tray medial surface, the silica gel heating strip is at least one, inlay in bearing the tray groove bottom surface and for and bear the closed annular structure of tray coaxial distribution, ultrasonic oscillation mechanism inlays in bearing the tray lateral wall surface to encircle bearing the tray axis equipartition, control circuit inlays in bearing the frame surface, and respectively with temperature sensor, check valve, ultrasonic oscillation mechanism, silica gel heating strip and revolving stage mechanism electrical connection.

A screening method for rapidly and primarily screening mycoplasma in a polluted milk sample comprises the following steps;

s1, pretreating milk, taking 1-5 ml as a milk sample, centrifuging at least one milk sample by a centrifuge, filtering the centrifuged milk sample by a filter at 0.40-0.45 mu m, and collecting filtrate for later use;

and S2, culturing and detecting, namely adding the filtrate collected in the step S1 and a liquid culture medium into a culture dish for culturing, and detecting by a color development method through a colorimetric detector after culturing for 20-60 minutes to obtain a mycoplasma detection result.

Further, the liquid culture medium comprises the following components in parts by weight: 10% -15% of PPLO broth, 5% -11% of compound horse serum, 13% -21% of 25% yeast extract, 2.3% -4.5% of ampicillin, 2.1% -3.5% of vancomycin, 3% -6% of glucose, 0.5% -1.8% of sodium pyruvate, 1.5% -3.1% of bovine lung digest, 0.3% -2.3% of 1% of phenol red, 0-11% of compound Erie's medium and the balance of ultrapure water.

Furthermore, the ampicillin is 5-8 ten thousand units.

Further, the preparation method of the 25% yeast extract comprises the following steps:

dissolving fresh yeast in ultrapure water with the mass 1.5-5 times that of the fresh yeast, uniformly stirring, fully dissolving the fresh yeast, boiling for 15min, quickly cooling to the normal temperature at the speed of 10-20 ℃/min, centrifugally separating cooling liquid at the speed of 3500-5000 r/min for 15min, extracting supernatant, boiling the supernatant again for 15min, quickly cooling to the normal temperature at the speed of 10-20 ℃/min, filtering by using a K-type clarifying filter, filtering by using a 0.22-micron sterile filter membrane for sterilization, taking filtrate, and storing the filtrate at the constant temperature of 4 ℃.

Further, the preparation method of the bovine lung digestive juice comprises the following steps:

firstly, boiling cattle lungs, crushing healthy cattle lungs, enabling the particle size of cattle lung particles to be not more than 1 mm, mixing the cattle lung particles with ultrapure water with the mass 2.5-6 times of that of the cattle lung particles, heating the mixed solution on an electric furnace to 60-80 ℃, and continuously stirring;

secondly, preparing a mixture, namely heating and preserving heat for 1-10 minutes, adding sodium carbonate accounting for 0.3-1.1% of the total amount of the mixed solution into the mixed solution, continuously preserving heat and stirring for at least 1 minute, then cooling to 45 ℃, adding a pancreatin solution accounting for 0.8-2.1% of the total amount of the mixed solution into the mixed solution again, finally carrying out water bath on the mixed solution for 3 hours at the constant temperature of 45 ℃, removing grease on the liquid level in the water bath process, and simultaneously adding concentrated hydrochloric acid accounting for 0.9-1.8% of the total amount of the mixed solution into the mixed solution and uniformly stirring;

and thirdly, filtering and collecting, boiling the mixed liquor prepared in the second step for 15min, cooling to normal temperature, filtering by double-layer gauze, centrifugally separating the filtrate for 15min at the speed of 2500-4000 r/min, collecting the filtrate, filtering the filtrate by using filter paper, boiling the filtrate filtered by the filter paper for 15min, cooling to room temperature, adjusting the pH value of the mixed liquor to 7.8 by using NaOH solution, sterilizing at 121 ℃ for 15min, and storing at constant temperature of 4 ℃.

Further, the preparation method of the liquid culture medium comprises the following steps:

the first step, the preparation treatment, namely, fully mixing PPLO broth, sodium pyruvate, MEM, bovine lung digestive juice, 1% phenol red and ultrapure water, adjusting the pH value to 7.8, then sterilizing at 121 ℃ for 15min, and cooling to 50-55 ℃;

and secondly, performing secondary treatment, namely adding the compound horse serum, the yeast leaching solution, ampicillin and vancomycin into the mixed solution prepared in the first step, uniformly mixing, and then regulating the pH value of the mixed solution to 7.8 to obtain a finished product.

According to the invention, the liquid culture medium is used for culturing the pretreated milk sample, and the experimental result is indicated through the chromogenic reaction, so that compared with the traditional detection method, the detection efficiency is greatly improved, the detection cost is reduced, the purposes of rapidly screening pathogenic pathogens and preliminarily diagnosing infectious bovine mastitis are achieved, the working cost and labor intensity of preliminary screening of milk in a pasture or a breast enterprise are effectively reduced, suspicious milk samples can be found in time, and the precision and efficiency of fresh milk quality detection are improved.

Drawings

The invention is described in detail below with reference to the drawings and the detailed description;

FIG. 1 is a schematic diagram of the system of the present invention;

FIG. 2 is a schematic view of the structure of a culture dish;

FIG. 3 is a flow chart of a screening method;

FIG. 4 is a flow chart of a method for preparing bovine lung digestive juice;

FIG. 5 is a schematic flow chart of a method for preparing a liquid medium.

The reference numbers in the figures: the device comprises a centrifuge 1, a filter 2, a culture dish 3, a chromaticity detector 4, a rotary table mechanism 5, a liquid filling port 6, an exhaust port 7, an elastic connecting mechanism 8, a sliding chute 9, a bearing frame 31, a bearing tray 32, a culture tray 33, a transparent sealing cover 34, a temperature sensor 35, a one-way valve 36, an ultrasonic oscillation mechanism 37, a silica gel heating strip 38, a control circuit 39, a bearing spring 321, an elastic cushion block 322 and a positioning groove 323.

Detailed Description

In order to facilitate the implementation of the technical means, creation features, achievement of the purpose and the efficacy of the invention, the invention is further described below with reference to specific embodiments.

As shown in fig. 1, a system for rapidly and primarily screening mycoplasma in a contaminated milk sample comprises a centrifuge 1, a filter 2, culture dishes 3 and a chromaticity detector 4, wherein the centrifuge 1 is connected with at least one filter 2 through a flow guide pipe, the filter 2 is further connected with a plurality of culture dishes 3, the culture dishes 3 are connected in parallel, the chromaticity detector 4 is located above the culture dishes 3, and an included angle of 30-90 degrees is formed between an optical axis of the chromaticity detector and the upper ends of the culture dishes 3.

Referring to fig. 2, the culture dish 3 comprises a bearing frame 31, a bearing tray 32, a culture disc 33, a transparent sealing cover 34, a temperature sensor 35, a one-way valve 36, an ultrasonic oscillation mechanism 37, a silica gel heating strip 38 and a control circuit 39, wherein the bearing frame 31 is a columnar frame structure with an axis vertical to the horizontal plane, the bearing tray 32 is a closed ring structure with a cross section in a shape of "u", is embedded in the upper end surface of the bearing frame 31 and is coaxially distributed with the bearing frame 31, the culture disc 33 is embedded in the upper end surface of the bearing tray 32 and is coaxially distributed against the bottom of the bearing tray 32, the culture disc 33 and the transparent sealing cover 34 are both in a structure with a cross section in a shape of "u" groove, the transparent sealing cover 34 covers the upper end surface of the culture disc 33 and forms a closed cavity structure with the culture disc 33, and the side surface of the transparent sealing cover 34 is hinged to the upper end surface of the bearing frame 31 through a turntable mechanism 5, a liquid filling opening 6 and an exhaust opening 7 are arranged on the transparent sealing cover 34, the liquid filling opening 6 and the exhaust opening 7 are symmetrically distributed on two sides of the axis of the culture tray 33 and form an included angle of 0-60 degrees with the axis of the culture tray 33, the liquid filling opening 6 and the exhaust opening 7 are both connected with a one-way valve 36, the temperature sensor 35 is embedded in the bearing tray 32 and is coaxially distributed with the bearing tray 32 and is connected with the inner side surface of the bearing tray 32 through an elastic connecting mechanism 8, at least one silica gel heating strip 38 is embedded on the upper surface of the groove bottom of the bearing tray 32 and is of a closed annular structure coaxially distributed with the bearing tray 32, the ultrasonic oscillation mechanism 37 is embedded on the outer surface of the side wall of the bearing tray 32 and is uniformly distributed around the axis of the bearing tray 32, the control circuit 39 is embedded on the outer surface of the bearing frame 31 and is respectively connected with the temperature sensor 35, the one-way valve 36, the ultrasonic oscillation mechanism 37, the temperature sensor 35, the exhaust opening 7 and the temperature sensor 32, The silica gel heating strip 38 is electrically connected with the turntable mechanism 5.

Meanwhile, the carrying tray 32 is embedded in the upper end surface of the carrying frame 31, and the outer side surface thereof is slidably connected with the inner side surface of the carrying frame 31 through the chute 9.

In addition, a plurality of bearing springs 321 and elastic cushion blocks 322 connected with the upper end faces of the bearing springs 321 are uniformly distributed on the upper end face of the bottom of the bearing tray 32 around the axis of the bearing tray 32, the axis of the bearing springs 321 is distributed in parallel with the axis of the bearing tray 32, the lower end face of the bearing springs 321 is connected with the bottom of the bearing tray 32, positioning grooves 323 coaxially distributed with the bearing springs 321 are arranged on the lower end face of the bearing tray 32 corresponding to the bearing springs 321, and the length of the bearing springs 321, the lower half parts of which are embedded in the positioning grooves 323 and located in the positioning grooves 323, is not less than 10% of the length of the bearing springs 321.

Example 1

Referring to fig. 3, a screening method for fast and primarily screening mycoplasma in a polluted milk sample comprises the following steps;

s1, pretreating the milk, taking 1 as a milk sample, centrifuging at least one milk sample by a centrifuge 1, filtering the centrifuged milk sample by a filter 2 at 0.40-mum, and collecting the filtrate for later use;

and S2, culturing and detecting, namely adding the filtrate collected in the step S1 and a liquid culture medium into a culture dish 3 for culturing, and detecting by a color development method through a colorimetric detector 4 after culturing for 20 minutes to obtain a mycoplasma detection result.

In this embodiment, the liquid medium is composed of the following components in parts by weight: 10% of PPLO broth, 5% of compound horse serum, 13% of 25% yeast extract, 2.3% of ampicillin, 2.1% of vancomycin, 3% of glucose, 0.5% of sodium pyruvate, 1.5% of bovine lung digestive juice, 0.3% of 1% of phenol red and the balance of ultrapure water.

Wherein, the ampicillin is 5 ten thousand units.

Notably, the preparation method of the 25% yeast extract comprises the following steps:

dissolving fresh yeast in ultrapure water with the mass 1.5-5 times of that of the fresh yeast, uniformly stirring, fully dissolving the fresh yeast, boiling for 15min, quickly cooling to the normal temperature at the speed of 10 ℃/min, centrifugally separating cooling liquid for 15min at 3500r/min, extracting supernatant, boiling the supernatant again for 15min, quickly cooling to the normal temperature at the speed of 10 ℃/min, filtering by using a K-type clarifying filter, filtering by using a 0.22-micron sterile filter membrane for sterilization, taking filtrate, and storing the filtrate at the constant temperature of 4 ℃.

Referring to fig. 4, the preparation method of the bovine lung digestive juice comprises the following steps:

firstly, boiling cattle lungs, namely smashing healthy cattle lungs, enabling the particle size of cattle lung particles to be not more than 1 mm, mixing the cattle lung particles with ultrapure water with the mass 2.5 times that of the cattle lung particles, heating the mixed solution on an electric furnace to 60 ℃, and continuously stirring;

secondly, preparing a mixture, namely heating the mixed solution prepared in the first step, keeping the temperature for 1-10 minutes, adding sodium carbonate accounting for 0.3% of the total amount of the mixed solution into the mixed solution, keeping the temperature and stirring for at least 1 minute, then cooling to 45 ℃, adding a pancreatin solution accounting for 0.8% of the total amount of the mixed solution into the mixed solution again, finally carrying out water bath on the mixed solution for 3 hours at the constant temperature of 45 ℃, removing oil on the liquid surface in the water bath process, and simultaneously adding concentrated hydrochloric acid accounting for 0.9% of the total amount of the mixed solution into the mixed solution and stirring uniformly;

and thirdly, filtering and collecting, boiling the mixed liquor prepared in the second step for 15min, cooling to normal temperature, filtering by double-layer gauze, centrifugally separating the filtrate for 15min at the speed of 2500 r/min, collecting the filtrate, filtering the filtrate by using filter paper, boiling the filtrate filtered by the filter paper for 15min, cooling to room temperature, adjusting the pH value of the mixed liquor to 7.8 by using NaOH solution, sterilizing at 121 ℃ for 15min, and storing at constant temperature of 4 ℃.

Referring to fig. 5, the preparation method of the liquid medium comprises:

the first step, the preparation treatment, namely, fully mixing PPLO broth, sodium pyruvate, MEM, bovine lung digestive juice, 1% phenol red and ultrapure water, adjusting the pH value to 7.8, then sterilizing at 121 ℃ for 15min, and cooling to 50-55 ℃;

and secondly, performing secondary treatment, namely adding the compound horse serum, the yeast leaching solution, ampicillin and vancomycin into the mixed solution prepared in the first step, uniformly mixing, and then regulating the pH value of the mixed solution to 7.8 to obtain a finished product.

Example 2

Referring to fig. 3, a screening method for rapidly and primarily screening mycoplasma in a polluted milk sample comprises the following steps;

s1, pretreating the milk, taking 5ml as a milk sample, centrifuging at least one milk sample by a centrifuge 1, filtering the centrifuged milk sample by a filter 2 at 0.45 mu m, and collecting the filtrate for later use;

and S2, culturing and detecting, namely adding the filtrate collected in the step S1 and a liquid culture medium into a culture dish 3 for culturing, and detecting by a color development method through a colorimetric detector 4 after culturing for 60 minutes to obtain a mycoplasma detection result.

Wherein the liquid culture medium comprises the following components in parts by weight: the feed comprises 15% of PPLO broth, 11% of compound horse serum, 21% of 25% yeast extract, 4.5% of ampicillin, 3.5% of vancomycin, 6% of glucose, 1.8% of sodium pyruvate, 3.1% of bovine lung digest and 2.3% of 1% phenol red, 11% of compound Erie's medium and the balance of ultrapure water, wherein the ampicillin is 8 ten thousand units.

In this embodiment, the preparation method of the 25% yeast extract solution includes:

dissolving fresh yeast in ultrapure water with the mass 5 times that of the fresh yeast, uniformly stirring, fully dissolving the fresh yeast, boiling for 15min, rapidly cooling to normal temperature at the speed of 20 ℃/min, centrifugally separating the cooling liquid for 15min at 5000r/min, extracting supernatant, boiling the supernatant again for 15min, rapidly cooling to normal temperature at the speed of 20 ℃/min, filtering by using a K-type clarifying filter, filtering by using a 0.22um sterile filter membrane for sterilization, taking filtrate, and storing the filtrate at the constant temperature of 4 ℃.

Referring to fig. 4, the preparation method of the bovine lung digestive juice comprises the following steps:

firstly, boiling cattle lungs, crushing healthy cattle lungs, enabling the particle size of cattle lung particles to be not more than 1 mm, mixing the cattle lung particles with ultrapure water with the mass 6 times that of the cattle lung particles, heating the mixed solution on an electric furnace to 80 ℃, and continuously stirring;

secondly, preparing a mixture, namely heating the mixed solution prepared in the first step, keeping the temperature for 1-10 minutes, adding sodium carbonate accounting for 1.1% of the total amount of the mixed solution into the mixed solution, keeping the temperature and stirring for at least 1 minute, then cooling to 45 ℃, adding a pancreatin solution accounting for 2.1% of the total amount of the mixed solution into the mixed solution again, finally carrying out water bath on the mixed solution for 3 hours at the constant temperature of 45 ℃, removing oil on the liquid surface in the water bath process, and simultaneously adding concentrated hydrochloric acid accounting for 1.8% of the total amount of the mixed solution into the mixed solution and stirring uniformly;

and thirdly, filtering and collecting, boiling the mixed liquor prepared in the second step for 15min, cooling to normal temperature, filtering by double-layer gauze, centrifugally separating the filtrate for 15min at the speed of 4000r/min, collecting the filtrate, filtering the filtrate by using filter paper, boiling the filtrate filtered by the filter paper for 15min, cooling to room temperature, adjusting the pH value of the mixed liquor to 7.8 by using NaOH solution, sterilizing at 121 ℃ for 15min, and storing at constant temperature of 4 ℃.

Referring to FIG. 5, in this example, the liquid medium preparation method is:

the first step, the preparation treatment, namely, fully mixing PPLO broth, sodium pyruvate, MEM, bovine lung digestive juice, 1% phenol red and ultrapure water, adjusting the pH value to 7.8, then sterilizing at 121 ℃ for 15min, and cooling to 50-55 ℃;

and secondly, performing secondary treatment, namely adding the compound horse serum, the yeast leaching solution, ampicillin and vancomycin into the mixed solution prepared in the first step, uniformly mixing, and then regulating the pH value of the mixed solution to 7.8 to obtain a finished product.

Example 3

Referring to fig. 3, a screening method for rapidly and primarily screening mycoplasma in a polluted milk sample comprises the following steps;

s1, pretreating milk, taking 2ml as a milk sample, centrifuging at least one milk sample by a centrifuge 1, filtering the centrifuged milk sample by a filter 2 at 0.43 mu m, and collecting filtrate for later use;

and S2, culturing and detecting, namely adding the filtrate collected in the step S1 and a liquid culture medium into a culture dish 3 for culturing, and detecting by a color development method through a colorimetric detector 4 after culturing for 30 minutes to obtain a mycoplasma detection result.

Wherein the liquid culture medium comprises the following components in parts by weight: 12% of PPLO broth, 8% of compound horse serum, 15% of 25% yeast extract, 3.5% of ampicillin, 2.5% of vancomycin, 4.1% of glucose, 0.8% of sodium pyruvate, 2.1% of bovine lung digestive juice, 1.3% of phenol red and the balance of ultrapure water.

Further optimized, the ampicillin is 6 ten thousand units.

It is emphasized that the preparation method of the 25% yeast extract comprises the following steps:

dissolving fresh yeast in ultrapure water with the mass 2.5 times that of the fresh yeast, uniformly stirring, fully dissolving the fresh yeast, boiling for 15min, rapidly cooling to normal temperature at the speed of 15 ℃/min, centrifugally separating the cooling liquid for 15min at the speed of 4000r/min, extracting supernatant, boiling the supernatant again for 15min, rapidly cooling to normal temperature at the speed of 15 ℃/min, filtering by using a K-type clarifying filter, filtering by using a 0.22um sterile filter membrane for sterilization, taking filtrate, and storing the filtrate at the constant temperature of 4 ℃.

Referring to fig. 4, the preparation method of the bovine lung digestive juice comprises the following steps:

boiling the cattle lungs, namely smashing healthy cattle lungs, enabling the particle size of cattle lung particles to be not more than 1 mm, mixing the cattle lung particles with ultrapure water with the mass 3 times that of the cattle lung particles, heating the mixed solution on an electric furnace to 70 ℃, and continuously stirring;

secondly, preparing a mixture, namely heating and preserving heat of the mixed solution prepared in the first step for 5 minutes, adding sodium carbonate accounting for 0.8 percent of the total amount of the mixed solution into the mixed solution, keeping the heat and stirring for at least 1 minute, then cooling to 45 ℃, adding a pancreatin solution accounting for 1.5 percent of the total amount of the mixed solution into the mixed solution again, finally carrying out water bath on the mixed solution for 3 hours at the constant temperature of 45 ℃, removing grease on the liquid surface in the water bath process, and simultaneously adding concentrated hydrochloric acid accounting for 1.3 percent of the total amount of the mixed solution into the mixed solution and stirring uniformly;

and thirdly, filtering and collecting, boiling the mixed liquor prepared in the second step for 15min, cooling to normal temperature, filtering by double-layer gauze, centrifugally separating the filtrate for 15min at 3000 r/min, collecting the filtrate, filtering the filtrate by using filter paper, boiling the filtrate filtered by the filter paper for 15min, cooling to room temperature, adjusting the pH value of the mixed liquor to 7.8 by using NaOH solution, sterilizing at 121 ℃ for 15min, and storing at constant temperature of 4 ℃.

Referring to FIG. 5, in this example, the liquid medium preparation method is:

the first step, the preparation treatment, namely, fully mixing PPLO broth, sodium pyruvate, MEM, bovine lung digestive juice, 1% phenol red and ultrapure water, adjusting the pH value to 7.8, then sterilizing at 121 ℃ for 15min, and cooling to 50-55 ℃;

and secondly, performing secondary treatment, namely adding the compound horse serum, the yeast leaching solution, ampicillin and vancomycin into the mixed solution prepared in the first step, uniformly mixing, and then regulating the pH value of the mixed solution to 7.8 to obtain a finished product.

Example 4

Referring to fig. 3, a screening method for rapidly and primarily screening mycoplasma in a polluted milk sample comprises the following steps;

s1, pretreating the milk, taking 3ml as a milk sample, centrifuging at least one milk sample by a centrifuge 1, filtering the centrifuged milk sample by a filter 2 at 0.43 mu m, and collecting the filtrate for later use;

and S2, culturing and detecting, namely adding the filtrate collected in the step S1 and a liquid culture medium into a culture dish 3 for culturing, and detecting by a color development method through a colorimetric detector 4 after culturing for 40 minutes to obtain a mycoplasma detection result.

The liquid culture medium is mainly characterized by comprising the following components in parts by weight: 12% of PPLO broth, 8% of compound horse serum, 14% of 25% yeast extract, 3.1% of ampicillin, 2.5% of vancomycin, 4.1% of glucose, 1.4% of sodium pyruvate, 2.1% of bovine lung digestive juice, 2.1% of phenol red and the balance of ultrapure water.

In this example, the ampicillin unit is 6 ten thousand units.

It is emphasized that the preparation method of the 25% yeast extract comprises the following steps:

dissolving fresh yeast in ultrapure water with the mass 3 times that of the fresh yeast, uniformly stirring, fully dissolving the fresh yeast, boiling for 15min, quickly cooling to normal temperature at the speed of 20 ℃/min, centrifugally separating the cooling liquid for 15min at 4000r/min, extracting supernatant, boiling the supernatant again for 15min, quickly cooling to normal temperature at the speed of 15 ℃/min, filtering by using a K-type clarifying filter, filtering by using a 0.22um sterile filter membrane for sterilization, taking filtrate, and storing the filtrate at the constant temperature of 4 ℃.

The yeast leaching solution prepared by the method has the advantages that the solubility of fresh yeast is increased by even stirring, the leaching solution filtered by the K-type clarifying filter is light yellow, the interference on the turbidity of a culture medium is avoided as much as possible, and the damage to the components of the leaching solution by a high-temperature and high-pressure sterilization method is avoided by using a 0.22-micron sterile filter membrane for filtration and sterilization.

Referring to fig. 4, the preparation method of the bovine lung digestive juice comprises the following steps:

boiling the cattle lungs, namely smashing healthy cattle lungs, enabling the particle size of cattle lung particles to be not more than 1 mm, mixing the cattle lung particles with ultrapure water with the mass 4.5 times that of the cattle lung particles, heating the mixed solution on an electric furnace to 70 ℃, and continuously stirring;

secondly, preparing a mixture, namely heating and preserving heat of the mixed solution prepared in the first step for 5 minutes, adding sodium carbonate accounting for 0.8 percent of the total amount of the mixed solution into the mixed solution, keeping the heat and stirring for at least 1 minute, then cooling to 45 ℃, adding a pancreatin solution accounting for 1.1 percent of the total amount of the mixed solution into the mixed solution again, finally carrying out water bath on the mixed solution for 3 hours at the constant temperature of 45 ℃, removing grease on the liquid surface in the water bath process, and simultaneously adding concentrated hydrochloric acid accounting for 0.3 percent of the total amount of the mixed solution into the mixed solution and stirring uniformly;

and thirdly, filtering and collecting, boiling the mixed liquor prepared in the second step for 15min, cooling to normal temperature, filtering by double-layer gauze, centrifugally separating the filtrate for 15min at 3000 r/min, collecting the filtrate, filtering the filtrate by using filter paper, boiling the filtrate filtered by the filter paper for 15min, cooling to room temperature, adjusting the pH value of the mixed liquor to 7.8 by using NaOH solution, sterilizing at 121 ℃ for 15min, and storing at constant temperature of 4 ℃.

Referring to FIG. 5, in this example, the liquid medium preparation method is:

the first step, the preparation treatment, namely, fully mixing PPLO broth, sodium pyruvate, MEM, bovine lung digestive juice, 1% phenol red and ultrapure water, adjusting the pH value to 7.8, then sterilizing at 121 ℃ for 15min, and cooling to 53 ℃;

and secondly, performing secondary treatment, namely adding the compound horse serum, the yeast leaching solution, ampicillin and vancomycin into the mixed solution prepared in the first step, uniformly mixing, and then regulating the pH value of the mixed solution to 7.8 to obtain a finished product.

According to the invention, the liquid culture medium is used for culturing the pretreated milk sample, and the experimental result is indicated through the chromogenic reaction, so that compared with the traditional detection method, the detection efficiency is greatly improved, the detection cost is reduced, the purposes of rapidly screening pathogenic pathogens and preliminarily diagnosing infectious bovine mastitis are achieved, the working cost and labor intensity of preliminary screening of milk in a pasture or a breast enterprise are effectively reduced, suspicious milk samples can be found in time, and the precision and efficiency of fresh milk quality detection are improved.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.