阅读说明：本技术 一种方便快捷鉴定养殖场沙门氏菌的方法 (Method for conveniently and rapidly identifying salmonella in farm ) 是由 刘丹丹 唐代苹 石艳红 李启蒙 于 2021-07-28 设计创作，主要内容包括：本发明公开了一种方便快捷鉴定养殖场沙门氏菌的方法,属于细菌检测技术领域,该方便快捷鉴定养殖场沙门氏菌的方法,S1、实验材料准备,预先对所需材料进行杀菌消毒和校准作业,S2、环境样本、垫料样本和饲料样本的采样,对样本的增菌处理,随后采用ss培养基进行菌落接种培养,待观察和分析。结合采样的范围,以及最终的检测结果,当养殖场产生大量的沙门氏菌感染时,工作人员可以第一时间内了解到养殖场内的沙门氏菌是从饲料、垫料、环境和雏鸡自带的毒株,可以在第一时间内针对携带情况进行防疫措施,其次是在检测过程中,大量使用增菌措施,一方面提高测量精度,另外一方面可以结合样本的数量进行合并检测,提高检测的效率。(The invention discloses a method for conveniently and quickly identifying salmonella in a farm, which belongs to the technical field of bacteria detection, and comprises the steps of S1, preparing experimental materials, carrying out sterilization, disinfection and calibration operation on required materials in advance, S2, sampling an environmental sample, a padding sample and a feed sample, carrying out enrichment treatment on the sample, then carrying out colony inoculation culture by adopting a ss culture medium, and waiting for observation and analysis. Combining the scope of sampling, and ultimate testing result, when the plant produced a large amount of salmonella and infected, the staff can know in the very first time that the salmonella in the plant is from fodder, bedding and padding, environment and chick own virulent strain, can carry out the epidemic prevention measure to the situation of carrying in the very first time, secondly in the testing process, use the enrichment measure in a large number, improve measurement accuracy on the one hand, on the other hand can combine the detection in combination with the quantity of sample, improve the efficiency of detecting.)

1. A method for conveniently and quickly identifying salmonella in a farm is characterized by comprising the following steps:

s1, preparing experimental materials, and carrying out sterilization, disinfection and calibration operation on the required materials in advance;

s2, sampling an environment sample, a padding sample and a feed sample, performing enrichment treatment on the samples, then performing colony inoculation culture by adopting a ss culture medium, and waiting for observation and analysis;

s3, sampling and processing an organ sample, then performing enrichment processing, and performing colony inoculation culture by adopting a ss culture medium to be observed and analyzed;

s4, collecting a weak chick sample with a cotton swab in a cloaca of a breeding hen, performing enrichment treatment, and inoculating the enrichment solution to a colorless transparent center black colony to inoculate for salmonella chromogenic culture;

s5, carrying out chromogenic culture of the salmonella and observation on a ss culture medium plate, and selecting the purified bacterial liquid to carry out further molecular biological method for identifying the species of the salmonella.

2. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S1, the experimental environment is disinfected, the operation table is scrubbed by 75% alcohol, then the laboratory and the super clean bench are disinfected by ultraviolet rays for 30min, cotton swabs, tweezers, plates and normal saline are autoclaved at 121 ℃ for 20min, and sc liquid enrichment broth, ss culture medium and salmonella chromogenic culture medium are prepared in advance.

3. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S2, the feed sample is wiped and disinfected by 75% alcohol cotton balls with two hands, 10 g of the feed sample is put into 90 ml of SC enrichment solution, and the SC enrichment solution is put into a constant temperature shaking table with 37 ℃ and 100rpm for enrichment for 18-24 h.

4. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S2, the padding in the henhouse can adopt samples collected by a shoe cover method, a sterilized cotton swab is dipped in sterile normal saline to slightly wet a cotton ball of the cotton swab, one side of the cotton swab where a shoe cover is sampled is coated, then the cotton swab is placed into sc enrichment fluid, 5 samples can be collected in one henhouse, the cotton swab enrichment is generally carried out by adopting a test tube, and the samples are cultured in a 37 ℃ incubator for 18-24 hours.

5. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S2, the environment sample is wiped and disinfected by 75% alcohol cotton balls on both hands, a sterilized cotton swab is dipped in sterile normal saline to slightly wet the cotton ball of the cotton swab, one side of the shoe cover to be sampled is coated, then the cotton swab is placed into sc enrichment fluid, 5 samples are collected according to the samples to be detected, the cotton swab enrichment is generally carried out by adopting a test tube, and the samples are cultured in an incubator at 37 ℃ for 18-24 hours.

6. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S2, the enrichment liquid of the environmental sample, the padding sample and the feed sample is streaked and inoculated on a ss culture medium, and the ss culture medium is cultured in an incubator at 37 ℃ for 22 to 24 hours, and then the colorless transparent black colony in the center is inoculated on a salmonella color development culture medium for 22 to 24 hours.

7. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S3, the organ samples are liver and yolk, the two hands are wiped and disinfected by 75% alcohol cotton balls, the surfaces of the organs are treated by the alcohol cotton balls, the organs are cut by using a sterile scalpel, a small amount of tissue homogenate is aseptically taken and put into 3ml of sc enrichment fluid, and the sc enrichment fluid is cultured in a 37 ℃ incubator for 18-24 hours; taking the enrichment fluid, streaking the enrichment fluid on a ss culture medium, culturing the enrichment fluid in a 37 ℃ incubator for 22-24 hours, inoculating a colorless transparent black center colony on a salmonella chromogenic culture medium, and culturing for 22-24 hours to obtain a result.

8. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operating procedure in S4, the cloaca cotton swab of the breeding hens: collecting cloaca of breeding hens by using a sterile cotton swab, putting the cloaca into 3ml of sc enrichment broth, and culturing in a 37 ℃ incubator for 18-24 hours; and (3) taking the enriched liquid, streaking and inoculating the enriched liquid on an ss culture medium, culturing in a 37 ℃ incubator for 22-24 hours, inoculating a colorless transparent black center colony on a salmonella chromogenic culture medium, culturing for 22-24 hours, and then observing the result, wherein the weak chick is detected by adopting the detection method in the step 3.

9. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the procedure in S5, the ss medium result judgment criteria are growth and colony color.

10. The method for conveniently and rapidly identifying salmonella in a farm according to claim 1, comprising the following steps: according to the operation steps in S5, the results of the Salmonella chromogenic medium are judged to be growth conditions and colony color.

Technical Field

The invention relates to the technical field of bacteria detection, in particular to a method for conveniently and quickly identifying salmonella in a farm.

Background

The bacterial antigen detection refers to a method for detecting whether corresponding antigens exist in a patient body by using known bacterial antibodies, and is used for auxiliary diagnosis of bacterial infectious diseases, such as detection of neisseria meningitidis, neisseria gonorrhoeae, streptococcus (streptococcus pyogenes, streptococcus agalactiae, streptococcus pneumoniae and the like), pathogenic escherichia coli, shigella, salmonella, legionella and the like.

The pathogens of salmonellosis, belonging to the family enterobacteriaceae, gram-negative enterobacteriaceae, are found in nearly one thousand species (or strains), which are classified into basic groups of bacteria such as A, B, C, D, E, etc. according to their antigenic components, among which there are mainly A paratyphoid in group A, B paratyphoid and Salmonella typhimurium in group B, C paratyphoid and cholera suis in group C, and T typhoid and enteritis in group D, etc. besides the diseases caused by B typhoid, B, and B, most of them can only cause diseases in animals such as domestic animals, mice and birds, but sometimes they can also contaminate the food of human to cause food poisoning.

At present, a farm for breeding chickens is usually used for detecting chickens and chickens died of diseases by adopting a timing spot check mode in order to prevent large-area infectious diseases, so that the death cause or the etiology is treated intensively, the infection scale is prevented from being enlarged, and secondly, when the breeding scale is enlarged, the detection efficiency is reduced due to the large detection quantity, the rescue time is further lost, and the infection scale cannot be controlled in time.

Disclosure of Invention

The invention aims to provide a method for conveniently and quickly identifying salmonella in a farm. The invention discloses a method for conveniently and quickly identifying salmonella in a farm, which combines the sampling range and the final detection result, when the farm is infected with a large amount of salmonella, workers can know that the salmonella in the farm is a strain from feed, padding, environment and chickens in the first time, and can perform epidemic prevention measures aiming at carrying conditions in the first time, and then uses a large amount of bacteria increasing measures in the detection process, so that the measurement precision is improved, the combined detection can be performed by combining the number of samples, and the detection efficiency is improved.

In order to achieve the above effects, the present invention provides the following technical solutions: a method for conveniently and quickly identifying salmonella in a farm comprises the following steps:

s1, preparing experimental materials, and carrying out sterilization, disinfection and calibration operation on the required materials in advance;

s2, sampling an environment sample, a padding sample and a feed sample, performing enrichment treatment on the samples, then performing colony inoculation culture by adopting a ss culture medium, and waiting for observation and analysis;

s3, sampling and processing an organ sample, then performing enrichment processing, and performing colony inoculation culture by adopting a ss culture medium to be observed and analyzed;

s4, collecting a weak chick sample with a cotton swab in a cloaca of a breeding hen, performing enrichment treatment, and inoculating the enrichment solution to a colorless transparent center black colony to inoculate for salmonella chromogenic culture;

s5, carrying out chromogenic culture of the salmonella and observation on a ss culture medium plate, and selecting the purified bacterial liquid to carry out further molecular biological method for identifying the species of the salmonella.

Further, according to the operation procedure in S1, the experimental environment is sterilized, the operation table is scrubbed by 75% alcohol, then the laboratory and the clean bench are sterilized by ultraviolet rays for 30min, cotton swabs, tweezers, plates and normal saline are sterilized by high pressure at 121 ℃ for 20min, and sc liquid enrichment medium, ss medium and salmonella chromogenic medium are prepared in advance.

Further, according to the operation procedure in S2, the feed sample is wiped and disinfected by 75% alcohol cotton balls with two hands, 10 g of the feed sample is put into 90 ml of SC enrichment solution, and the SC enrichment solution is put into a constant temperature shaker at 37 ℃ and 100rpm for enrichment for 18-24 h.

Further, according to the operation step in S2, the padding in the chicken house may be samples collected by a shoe cover method, a sterilized cotton swab is dipped in sterile physiological saline to slightly wet a cotton ball of the cotton swab, one surface of the cotton swab where the shoe cover is sampled is coated, and then the cotton swab is put into sc enrichment broth, one chicken house may collect 5 samples, the cotton swab enrichment generally adopts test tube enrichment, and the samples are cultured in an incubator at 37 ℃ for 18-24 hours.

Further, according to the operation procedure in S2, the environmental sample is wiped and disinfected with a 75% alcohol cotton ball by both hands, a sterilized cotton swab is dipped in sterile normal saline to slightly wet the cotton ball of the cotton swab, one side of the shoe cover to be sampled is coated, then the cotton swab is put into sc enrichment fluid, 5 samples are collected according to the samples to be detected, the cotton swab enrichment is generally performed by using a test tube, and the samples are cultured in an incubator at 37 ℃ for 18-24 hours.

Further, according to the operation procedure in S2, the enrichment solution of the environmental sample, the bedding sample and the feed sample is streaked and inoculated on the ss culture medium, and the ss culture medium is cultured in an incubator at 37 ℃ for 22 to 24 hours, and then the colorless transparent black colony in the center is inoculated on the salmonella chromogenic culture medium and cultured for 22 to 24 hours.

Further, according to the operation procedure in S3, the organ samples were liver and yolk, the hands were wiped with a 75% alcohol cotton swab, the organ surface was treated with an alcohol cotton swab, the organs were dissected with a sterile scalpel, a small amount of tissue homogenate was aseptically taken, placed in 3ml of sc enrichment broth, and incubated in a 37 ℃ incubator for 18-24 hours; and taking the enrichment liquid, streaking and inoculating the enrichment liquid on an ss culture medium, culturing the enrichment liquid in an incubator at 37 ℃ for 22-24 hours, inoculating a colorless transparent black center colony on a salmonella chromogenic culture medium, and culturing for 22-24 hours to obtain a result.

Further, according to the operation step in S4, the cloaca cotton swab of the breeding hens: collecting cloaca of breeding hens by using a sterile cotton swab, putting the cloaca into 3ml of sc enrichment broth, and culturing in a 37 ℃ incubator for 18-24 hours; and (3) taking the enriched liquid, streaking and inoculating the enriched liquid on an ss culture medium, culturing in a 37 ℃ incubator for 22-24 hours, inoculating a colorless transparent black center colony on a salmonella chromogenic culture medium, culturing for 22-24 hours, and then observing the result, wherein the weak chick is detected by adopting the detection method in the step 3.

Further, according to the operation in S5, the ss medium result judgment criteria are growth and colony color.

Further, according to the operation in S5, the salmonella chromogenic medium result judgment criteria are growth and colony color.

The invention provides a method for conveniently and quickly identifying salmonella in a farm, which has the following beneficial effects:

actually, the invention discloses a method for conveniently and quickly identifying salmonella in a farm, which combines the sampling range and the final detection result, when the farm is infected with a large amount of salmonella, workers can know that the salmonella in the farm is a strain carried by feed, padding, environment and chicks in the first time, and can perform epidemic prevention measures aiming at carrying conditions in the first time, and then in the detection process, a large amount of bacteria increasing measures are used, so that the measurement precision is improved, and the combined detection can be performed by combining the number of samples, and the detection efficiency is improved.

Drawings

FIG. 1 is a schematic diagram of the method for conveniently and rapidly identifying Salmonella in a farm according to the present invention;

FIG. 2 is a schematic diagram of the color development culture of Salmonella in the method for conveniently and rapidly identifying Salmonella in a farm.

Detailed Description

The invention provides a technical scheme that: referring to fig. 1-2, a method for conveniently and rapidly identifying salmonella in a farm comprises the following steps:

step one, experimental material preparation, and sterilization, disinfection and calibration operation are carried out on the required material in advance.

And step two, sampling an environment sample, a padding sample and a feed sample, performing enrichment treatment on the samples, then performing colony inoculation culture by adopting a ss culture medium, and observing and analyzing.

And step three, sampling and processing the organ sample, then performing enrichment processing, and performing colony inoculation culture by adopting a ss culture medium to be observed and analyzed.

And step four, collecting a brood sample of the cloaca cotton swab of the breeding hens, performing enrichment treatment, and then inoculating the enrichment liquid to a colorless transparent center black colony to inoculate the colony to salmonella for chromogenic culture.

And fifthly, carrying out color development culture on the salmonella and observing on a ss culture medium plate, and selecting the purified bacterium liquid to carry out specific molecular biological method for identifying the species of the salmonella.

Specifically, according to the operation steps in S1, the experimental environment is disinfected, the operation table is scrubbed by 75% alcohol, then the laboratory and the clean bench are disinfected by ultraviolet rays for 30min, cotton swabs, tweezers, plates and normal saline are autoclaved at 121 ℃ for 20min, and sc liquid enrichment broth, ss culture medium and salmonella chromogenic culture medium are prepared in advance.

Specifically, according to the operation step in S2, the feed sample is wiped and disinfected by 75% alcohol cotton balls with both hands, 10 g of the feed sample is put into 90 ml of SC enrichment solution, and the SC enrichment solution is put into a constant temperature shaker at 37 ℃ and 100rpm for enrichment for 18-24 h.

Specifically, according to the operation step in S2, the padding in the chicken house may be samples collected by a shoe cover method, a sterilized cotton swab is dipped in sterile physiological saline to slightly wet the cotton ball of the cotton swab, the sampled surface of the shoe cover is coated, and then the cotton ball is placed in sc enrichment broth, one chicken house can collect 5 samples, the enrichment of the cotton swab is generally performed by using a test tube, and the samples are cultured in a 37 ℃ incubator for 18-24 hours.

Specifically, according to the operation steps in S2, the environment sample is wiped and disinfected by 75% alcohol cotton balls on both hands, sterilized cotton swabs are dipped in sterile normal saline to slightly wet cotton balls of the cotton swabs, one side of a shoe cover to be sampled is coated, then the cotton swabs are placed into sc enrichment solution, 5 samples are collected according to the samples to be detected, the cotton swabs are generally enriched by test tubes, and the samples are cultured in an incubator at 37 ℃ for 18-24 hours.

Specifically, according to the operation steps in S2, enrichment liquid of an environmental sample, a bedding material sample and a feed sample is streaked and inoculated on an ss culture medium, and the ss culture medium is placed in an incubator at 37 ℃ for culture for 22-24 hours, and then a colorless transparent black center colony is inoculated on a salmonella color development culture medium for culture for 22-24 hours.

Specifically, according to the operation procedure in S3, the organ samples were liver and yolk, both hands were wiped with 75% alcohol cotton balls for sterilization, the alcohol cotton balls were used to treat the organ surfaces, the organs were cut with a sterile scalpel, a small amount of tissue homogenate was aseptically taken, placed in 3ml of sc enrichment broth, and cultured in a 37 ℃ incubator for 18-24 hours; and taking the enrichment liquid, streaking and inoculating the enrichment liquid on an ss culture medium, culturing the enrichment liquid in an incubator at 37 ℃ for 22-24 hours, inoculating a colorless transparent black center colony on a salmonella chromogenic culture medium, and culturing for 22-24 hours to obtain a result.

Specifically, according to the operation procedure in S4, the cloaca cotton swab of the breeding hens: collecting cloaca of breeding hens by using a sterile cotton swab, putting the cloaca into 3ml of sc enrichment broth, and culturing in a 37 ℃ incubator for 18-24 hours; and (3) taking the enrichment fluid, streaking and inoculating the enrichment fluid on an ss culture medium, culturing in a 37 ℃ incubator for 22-24 hours, inoculating a colorless transparent black center colony on a salmonella chromogenic culture medium, culturing for 22-24 hours, and then observing the result, wherein the weak chick is detected by adopting the detection method in the step 3.

Specifically, according to the procedure in S5, the ss medium result judgment criteria are growth and colony color.

Specifically, according to the operation procedure in S5, the results of the salmonella chromogenic medium are judged as the growth state and the colony color.

The method of the examples was performed for detection analysis and compared to the prior art to yield the following data:

	detection accuracy	Detect the comprehensiveness	Efficiency of detection
				Examples	Is higher than	Is higher than	Is higher than
Prior Art	Is lower than	Is lower than	Is lower than

Judging the plate result of the ss culture medium:

culturing at 37 ℃ for 22-24 h:

quality control strain	Growth conditions	Color of colony
			Escherichia coli	+	Red colour
Salmonella	++	Colorless transparent central black

Judging the result of the flat plate of the salmonella chromogenic medium:

culturing at 37 ℃ for 22-24 h:

quality control strain	Growth conditions	Color of colony
			Prunus simplex	-	-
Staphylococcus aureus	-	-
			Escherichia coli	+/++	Blue color
Salmonella	+++	Purple color
			Salmonella typhimurium	+++	Purple color
Proteus mirabilis	-	Colorless and colorless
			Pseudomonas aeruginosa	-	Pale purple

According to the table data, the method for conveniently and quickly identifying the salmonella in the farm comprises the following steps: step one, preparing experimental materials, sterilizing and disinfecting required materials in advance, calibrating, testing environment for disinfection, scrubbing an operation table by using 75% alcohol, disinfecting a laboratory and an ultra-clean table by using ultraviolet rays for 30min, sterilizing a cotton swab, a pair of tweezers, a plate and physiological saline at high pressure for 20min at 121 ℃, preparing an SC liquid enrichment medium, an ss culture medium and a salmonella chromogenic culture medium in advance, sampling an environmental sample, a padding sample and a feed sample, enriching the samples, inoculating and culturing bacterial colonies by using the ss culture medium, wiping and disinfecting the feed sample by using a 75% alcohol cotton ball with two hands to obtain a 10 g sample, putting the 10 g sample into 90 ml of SC enrichment medium, enriching for 18-24h before placing the SC enrichment medium in a constant temperature shaking table at 37 ℃ and 100rpm, collecting the padding in a henhouse by using a shoe cover method, dipping the sterilized cotton swab into sterile physiological saline to slightly wet the cotton ball, smearing the sampled surface of the shoe cover, then putting the shoe cover into sc enrichment solution, 5 samples can be collected from a henhouse, generally adopting test tubes to enrich the cotton swabs, culturing the cotton swabs in an incubator at 37 ℃ for 18-24h, wiping and disinfecting the environmental samples with 75% alcohol cotton balls by hands, dipping the sterilized cotton swabs into sterile normal saline to slightly wet the cotton balls of the cotton swabs, smearing the sampled surface of the shoe cover, then putting the cotton swabs into sc enrichment solution, adopting 5 sample combinations according to the samples to be detected, generally adopting test tubes to enrich the cotton swabs, culturing the cotton swabs in the incubator at 37 ℃ for 18-24h, inoculating the enrichment solution of the environmental samples, the padding samples and the feed samples onto ss culture medium, culturing the obtained enrichment solution in the incubator at 37 ℃ for 22-24h, inoculating colorless transparent black colonies in the center onto salmonella chromogenic culture medium, and sampling the organ samples, then, enrichment treatment is carried out, a ss culture medium is adopted for colony inoculation culture, after observation and analysis, organ samples are livers and egg yolks, hands are wiped and disinfected by 75% alcohol cotton balls, the surfaces of the organs are treated by the alcohol cotton balls, the organs are cut by using an aseptic scalpel, a small amount of tissue is aseptically taken and homogenized, the homogenate is put into 3ml of sc enrichment solution, and the sc enrichment solution is cultured in a 37 ℃ incubator for 18-24 hours; taking the enrichment fluid, streaking and inoculating the enrichment fluid on an ss culture medium, placing the culture medium in a 37 ℃ incubator for culturing for 22-24 hours, then inoculating a colorless transparent center black colony on a salmonella chromogenic culture medium for culturing for 22-24 hours, and then observing the result, and step four, collecting a chick sample from a cloaca cotton swab, firstly carrying out enrichment treatment, then inoculating the enrichment fluid on a colorless transparent center black colony inoculum salmonella chromogenic culture, and carrying out a cloaca cotton swab: collecting cloaca of breeding hens by using a sterile cotton swab, putting the cloaca into 3ml of sc enrichment broth, and culturing in a 37 ℃ incubator for 18-24 hours; and taking the enriched liquid, streaking and inoculating the enriched liquid on an ss culture medium, culturing in a 37 ℃ incubator for 22-24 hours, inoculating a colorless transparent center black colony on a salmonella chromogenic culture medium, culturing for 22-24 hours, and then observing a result, wherein the weak chick detection adopts a detection method in step 3, the salmonella chromogenic culture and the ss culture medium plate are observed, the purified bacterial liquid is selected for identifying the species of salmonella by a molecular biological method, the ss culture medium result judgment standard is the growth condition and the colony color, and the salmonella chromogenic culture medium result judgment standard is the growth condition and the colony color.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in this embodiment without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.