KR101617166B1 - Reporter phage specifically infecting Salmonella with bioluminescence and method for detection of Salmonella - Google Patents

Abstract

The present invention relates to a genetically engineered bacteriophage that specifically infects Salmonella bacteria to produce bioluminescence and a method for detecting salmonella using the bacteriophage, and can effectively detect Salmonella in food using the bacteriophage of the present invention as a reporter phage.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for detecting a salmonella in a genetically engineered reporter phage that specifically infects Salmonella bacteria and shows bioluminescence, and a method for detecting Salmonella using the same.

TECHNICAL FIELD The present invention relates to a genetically engineered bacteriophage that specifically infects salmonella bacteria and exhibits bioluminescence and a method for detecting salmonella using the same. More particularly, the present invention relates to a bacteriophage used as a reporter phage to identify salmonella through bioluminescence, And a method for detecting Salmonella using the same.

Salmonella typhimurium bacteria (Salmonella Typhimurium) is a rod of a movement having a peripheral flagella with gram-negative bacteria, it is a facultative anaerobic bacteria do not form spores. Ingestion of meat, eggs, milk and other foods contaminated with this bacterium can cause acute food poisoning with fever and diarrhea. This bacterium grows rapidly when attached to foods such as meat and makes toxins in the cells, but there is a problem that the deterioration of the food can not be known due to appearance, taste, and smell.

To detect salmonella contaminated with food, methods such as total bacterial count, PCR, and immunoassay have been used. However, the total number of bacteria can detect all possible bacteria, but it takes a comparatively long time (48 hours) and can not specifically detect Salmonella. There is PCR or immunoassay as a method to detect Salmonella in a relatively early time. When used in food, it may be less susceptible to false-positive results have.

In order to overcome this problem, bacteriophage can be used as a detection substance of Salmonella. After plant viruses and animal viruses were discovered, Fredetick Twort first observed staphylococci in 1915, and in 1917 Felix Derell, of the Pasteur Institute, observed the same phenomenon in fungi, (Bacterial viruses). Since then, a virus infecting a bacterium has been termed a bacteriophage in the sense of "phage" in the term "bacterium" and is simply referred to as phage.

The phage has a lytic cycle and a lysogenic cycle. The phage is called a virulent phage and the temperate phage is a phagocytic phage. . However, unlike toxic phages, which dissolve host bacteria and dissolve newly synthesized phages through replication, lyophilized fungi insert their own dielectrics into the genome of the host bacteria It replicates its own genome continuously with the propagation of host bacteria. This state of availability lasts for as long as the host bacteria are in good environmental conditions, and when the environment deteriorates, the host is transformed into a virulent state, where new phages are generated from the inserted phage gene and finally the host bacteria are lysed and released outside the host cell . In the case of warm-tempered phage, since the host can be maintained as a pharmacokinetic circle without dissolving the host immediately after the bacterial infection, it is useful to amplify the signal even more when the target host bacterium, for example, Salmonella, is detected.

Although phage is a bacterial virus that infects only bacteria, it has been a problem to cause moral disturbance to people in using it because it belongs to viruses. In addition, in the case of bacteriophage subjected to genetic manipulation, the use of phage was not free because of the concern that such a variant phage spread to the natural world.

However, if the food to be tested is brought into the laboratory and used in this genetically engineered reporter phage, or in the form of a small kit containing the reporter phage to act only within it, they will spread to the natural world and affect other organisms Disadvantages that can cause discomfort can be minimized.

There are many ways to make genetically engineered reporter phage to detect foodborne pathogens in food. Reporter genes are also used. It is mainly used by a lacZ, gfp (green fluorescence protein), luxAB genes, of which luxAB gene is the portion of the luxCDABE operon, to generate a luciferase (luciferase), in the presence of its substrate, patio aldehyde (fatty aldehyde) It represents bioluminescence. Advantages of luxAB reporter phage include high sensitivity and low background interference. Therefore, attempts have been made to detect various food poisoning bacteria by inserting the luxAB gene into diverse phage genomes.

However, since the luxAB gene can only produce luciferase, it is troublesome to add the fatty aldehyde, which is a substrate of luciferase, to the reaction solution for the luminescence reaction have. In addition, since the light emission is rapidly reduced after the treatment with the fatty aldehyde, the error can be largely varied according to the skill of the experimenter and the continuous light emission can not be observed.

Korean Patent Laid-Open No. 10-1995-7000404 (published on Jan. 16, 1995) discloses a reporter maiko bacteriophage specific for mycobacteria species.

In the present invention, a method for rapidly and accurately detecting the infection of Salmonella using bacteriophage is developed and provided.

The present invention is based on the int integrase, oac (O-antigen acetylase), gtrA (putative bactoprenol-linked glucose translocase) gene and cps a major capsid protein operon, wherein the int , oac , gtrA The gene was removed and luxCDABE The operon The present invention provides a bacteriophage characterized by being inserted.

Due to the structural nature of the bacteriophage, a large increase in its gene length does not allow the gene to be fully located within its hair structure. Therefore, conventional techniques have only to select luxAB in luxCDABE and insert it into bacteriophage.

In order to insert the luxCDABE operon of the present invention having a 6-kilo base pair size, it is necessary to delete a part of the existing gene existing in the phage. However, since the erasure of the untimely gene deletes the inherent characteristic of the bacteriophage, The selection of genes requires considerable technical considerations.

Thus, in the present invention, three genes, int integrase, oac (O-antigen acetylase), gtrA (putative bactoprenol-linked glucose transglucose) gene was selected, and it was found that the deletion of the bactoprenol-linked glucose transglucose gene had no significant effect on the usability and the like, and the present invention was completed. That is, it is confirmed that the bacteriophage can be produced in the present invention without destroying the purpose of using the bacteriophage even when three genes are deleted.

Since the present invention has inserted the luxCDABE operon as a whole, it can not only produce luciferase by luxAB , but also can be produced by the luxCDE -encoded fatty acid reductase, (1993). "Bacterial bioluminescence: organization, regulation, and application of the lux genes." FASEB J 7 (1993) 11): 1016-1022. Therefore, only living Salmonella can be specifically detected without addition of a specific substrate.

In the bacteriophage of the present invention, the luxCDABE operon is preferably a bacteriophage cps (major capsid protein) operon, so that transcription is regulated by the cps promoter.

In the bacteriophage of the present invention, the bacteriophage may be, for example, SPC32H :: lux CDABE (KCCM 11441P).

Meanwhile, the present invention provides a salmonella detection method, wherein the bacteriophage of the present invention is used to detect Salmonella from a food.

In the salmonella detection method of the present invention, the salmonella detection method can measure the bioluminescence after infecting the bacteriophage of the present invention to a food infected with Salmonella, for example.

In the salmonella detection method of the present invention, the salmonella detection method can measure the bioluminescence using various methods. For example, the measurement can be performed using a luminometer.

In the salmonella detection method of the present invention, the food may be applied to any food to which Salmonella can be infected, for example, meat or milk.

The bacteriophage (reporter phage) of the present invention does not require the treatment of any additional material, unlike conventional reporter phages based on luxAB , which needed to additionally add a fatty aldehyde as substrate. As a result, the detection process is simpler since the bioluminescence can be measured immediately after infection with the reporter phage without any further treatment.

In addition, since the reporter phage of the present invention exhibits uniform bioluminescence in the cells upon infection with the host Salmonella, the error of the experiment is greatly reduced in the step of measuring the bioluminescence. Therefore, the reporter phage of the present invention is improved in convenience and accuracy as compared with existing reporter phage.

In addition, the detection of bacteria using the reporter phage of the present invention is based on the bioluminescence using the lux gene. In view of the fact that most foods do not exhibit bioluminescence, background interference There is almost no advantage. Therefore, the reporter phage of the present invention specifically detects Salmonella in a meat-processed food sample such as processed ham, which is difficult to specifically detect a target bacterium by PCR or immunoassay, which is a conventional detection method Lt; / RTI >

In addition, the reporter phage of the present invention decreased the intensity of bioluminescence by 10 times when used in milk, which is a sample of turbid liquid food, but the number of Salmonella bacteria present in the sample was linearly proportional to the intensity of bioluminescence Therefore, even a turbid liquid food sample such as milk can be used to specifically detect Salmonella.

1 is an electron micrograph of bacteriophage SPC32H used in the present invention.
Fig. 2 is a schematic diagram showing the production process of SPC32H :: luxCDABE . (A) shows the deletion gene and the introduction part of luxCDABE operon using λ Red recombination method, and (B) shows the introduction process of luxCDABE operon sequentially using In-Out method.
FIG. 3 shows the results of evaluating the reduction of the SPC32H :: luxCDABE for Salmonella.
Fig. 4 shows the results of measurement of salmonella detection ability of SPC32H :: lux CDABE .
Fig. 5 shows the results of the detection of SPC32H :: luxCDABE (A: lettuce, B: processed ham, C: milk) in artificially contaminated salmonella.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  1: Bacteriophage used in the present invention SPC32H  Separation and identification]

 (1) Isolation of bacteriophage

In order to isolate bacteriophages that specifically infect Salmonella, a fecal sample of chicken was taken from the Moran market located in Seongnam city, Gyeonggi - do and bacteriophage separation experiments were conducted therefrom.

25 g of the sample was mixed with Butterfield's phosphate-buffered dilution water and homogenized. Then, 10 mL of the homogeneous solution was mixed with 50 mL of liquid nutrient broth (LB broth).

The mixture was incubated at 37 ° C for 24 hours, then centrifuged at a rate of 9000 x g to take the supernatant, and then filtered through a 0.22 μm membrane filter. 10 mL of the filtrate and 0.1 mL of the salmonella culture solution cultured for 12 hours or more were mixed with 50 mL of a liquid nutrient medium and cultured at 37 ° C. for 8 hours, followed by centrifugation and filtration as described above. 0.1 mL of this filtrate was mixed with the same amount of Salmonella culture medium and 0.4% concentration of agar liquid medium and poured into a plate containing 1.5% agar solid medium.

The plate was then incubated overnight at 37 < 0 > C. When the desired bacteriophage was present in the sample, an independent transparent clear plaque was observed on the plate. This plated region was taken by using a sterilized platinum loop and then cultured at 37 ° C in a fresh salmonella culture. As a result, it was observed that the bacterial culture solution became clear and the cell debris accumulated.

The bacteriophage solution obtained by the above procedure was diluted and injected differently on the agar medium to perform re-separation of the lytic group three times or more. Through this series of steps, pure bacteriophage was obtained.

 (2) Observation and classification of morphology of bacteriophage SPC32H

The bacteriophage obtained in (1) above was named SPC32H and the morphology of the phage particle was analyzed by electron microscope. Transmission electron microscopy (TEM) was used for the electron microscope. The bacteriophage sample to be observed was placed on a carbon-coated copper grid and negatively stained with 2% liquid uranyl acetate. The dyed samples were observed with a transmission electron microscope (LIBRA 120, Carl Zeiss) at a voltage of 120 kV and the results are shown in FIG.

1 is an electron micrograph of bacteriophage SPC32H used in the present invention. When the particles of the bacteriophage observed in FIG. 1 are classified on the basis of their shape, the phage has a short, non-contractile tail on the isometric head, so that the podoviridae family). Grapevine band and bacteriophage obtained by the above method were named SPC32H.

[ Example  2: Reporter phage SPC32H :: luxCDABE  making]

To prepare the gene manipulated reporter phage SPC32H :: luxCDABE of the present invention, the host bacteria, S. By infection of the SPC32H Typhimurium LT2 (c) strain, S was inserted into the genome of SPC32H in their genome. Typhimurium LT2 (c) lysogen strain was obtained.

Due to its structural nature, bacteriophage can not fully position the gene in its hair structure if its gene length is greatly increased. Therefore, deletion of a part of the existing SPC32H gene is necessary for insertion of luxCDABE operon of 6-kilo base pair size. Therefore, three genes, int integrase, oac (O-antigen acetylase), gtrA (putative bactoprenol-linked glucose translocase) was deleted from the SPC32H genome.

Prok Natl Acad Sci USA 97 (12), " One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products ", Datsenko, KA and BL Wanner ): 6640-6645).

The luxCDABE operon cloned from pBBRlux followed by the cps (major capsid protein) operon of bacteriophage SPC32H deleted from the above three genes (Lenz, DH, et al. harveyi and Vibrio cholerae. "Cell 118 (1): 69-82.). Since the cps promoter of SPC32H may lead to strong expression, it may exhibit stronger bioluminescence after infection with Salmonella.

The in-out method for inserting the luxCDABE operon (Madyagol, M., et al. (2011). "Gene replacement techniques for Escherichia coli genome modification." Folia Microbiol (Praha) 56 (3): 253-263.) Was used. The cps promoter was used to transform luxCDABE The expression of operon was confirmed.

Fig. 2 is a schematic diagram showing the production process of SPC32H :: luxCDABE . (A) shows the deletion gene and the introduction part of luxCDABE operon using λ Red recombination method, and (B) shows the introduction process of luxCDABE operon sequentially using In-Out method.

The genetically engineered reporter phage SPC32H :: luxCDABE obtained by the above method was deposited on August 1, 2013 in KCCM, which is a microorganism depository institution, and received the accession number KCCM11441P.

[ Example  3: Reporter phage SPC32H :: luxCDABE Wow SPC32H Salmonella Drag force  compare]

The reporter phage of the present invention SPC32H ::luxCDABETo make Salmonella lysate experiments were conducted to test whether the genetic manipulation that was performed inhibited the original lytic ability of phage.

S. Typhimurium LT2 strain was cultured at 37 ° C and 220 rpm until OD ₆₀₀ value reached 0.5, and 50 mL of ⁴ × 10 ⁷ CFU / mL bacterial culture was obtained (CFU; colony forming units, One of the units representing the number of bacteria).

The reporter phage SPC32H :: luxCDABE , the original phage SPC32H, and the other intermediate stage phage were inoculated into a 50 mL salmonella strain (MOI; multiplicity of infection; usually mL ^-1 PFU / mL to a value showing the number of CFU of the bacteriophage to the ^{proportional-calculated} as PFU and ^1; plaque forming units, unit which forms the gyunban for the bacteriophage infects the bacteria, the unit indicating the number of phage) .

The flask was incubated at 37 ° C and 220 rpm, and the OD ₆₀₀ The values were measured with a spectrophotometer.

The OD ₆₀₀ value proportional to the time obtained from the experiment is shown in FIG. 32H shown in FIG. 3 is SPC32H, AB is SPC32H cps - luxAB , IO AB is SPC32H Δ int Δ oac cps - luxAB , IO CDABE is SPC32H Δ int Δ oac cps - luxCDABE respectively.

int In the case of the integrase gene, since it is a gene conferring virulence, the lytic characteristic was changed when the gene was deleted. Phages with the int gene rapidly formed Lysozene and increased the OD ₆₀₀ value. On the other hand, the phage in which the int gene was deleted retained the ability to form lysogen and the OD ₆₀₀ value remained low for a longer time. However, all of these phages exhibited a solubilizing power against Salmonella to a similar extent as the original SPC32H. Therefore, it was concluded that the genetic manipulation of the reporter phage of the present invention did not significantly affect the lytic activity of the SPC32H phage.

[ Example  4: Reporter phage SPC32H :: luxCDABE ≪ / RTI > Salmonella <

S. Typhimurium LT2 strain cultured in 37 ℃, 220 rpm until the OD ₆₀₀ value to be 0.5 4ⅹ10 ⁷ CFU / mL of the culture solution with the bacteria get around, and diluted sequentially by one-tenth in the liquid nutrient medium from 10 10 ⁵ CFU / mL.

Each dilution of the culture solution was transferred to a test tube in an amount of 5 mL, and a reporter phage SPC32H :: luxCDABE prepared at a concentration of 10 ⁸ PFU / mL was inoculated.

The inoculum concentration of 10 < ⁸ > PFU / mL is the minimum inoculum concentration obtained from the optimization experiment to give the maximum bioluminescence.

The inoculated test tubes were mixed well and incubated at 37 ° C and 220 rpm for 2 hours. Then, 200 μl of the culture was transferred to a tube for bioluminescence measurement, and the bioluminescence was measured for 10 seconds using a 'berthold luminometer', and the result was expressed as RLU (relative luminescence unit) value.

Fig. 4 shows the results of measurement of salmonella detection ability of SPC32H :: lux CDABE . SPC32H :: luxCDABE was able to detect at least 20 CFU / mL salmonella significantly, and it was confirmed that the number of salmonella increased linearly with increasing Salmonella count. .

[ Example  5: Reporter phage SPC32H :: luxCDABE Detection of artificially contaminated Salmonella in food using

 (1) Detection of salmonella bacteria contaminated with cabbage artificially

In order to test whether the reporter phage of the present invention can detect Salmonella contaminated with food, lettuce, processed ham, and milk were selected as model foods.

Fresh vegetable food lettuce ( Lactuca sativa L.) was consumed without the bacterial reduction process, so it was judged that the detection of Salmonella was necessary and selected as a model food. Lettuce sold in the market was cut and cut to a size of about 10 cm x 2 cm using a sterilized stainless steel knife and grouped to weigh 10 g. 10 mL of the diluted Salmonella culture prepared as in Example 4 was inoculated into lettuce weighing 10 g each using a pipette and then dried on a clean bench for 30 minutes. Each lettuce sample was then mixed with 90 mL of nutrient medium, homogenized using a stomacher, and plated on XLD medium (Xylose Lysine Deoxycholate), a selective medium for salmonella, to determine the number of salmonella actually inoculated per gram of lettuce Were measured. In addition, 5 mL of the homogenized sample was transferred to a sterilized test tube. In the same manner as in Example 3, a reporter phage was inoculated and cultured for 2 hours, and then the RLU value was measured using a luminometer.

The graph of FIG. 5 (A) was obtained based on the number of salmonella bacteria obtained by the above method and the RLU value. This graph is similar to the graph obtained by experiment with pure salmonella culture (Fig. 4). When the R ² value of the graph is close to 1, the number of salmonella present in the cabbage can be accurately estimated by measuring the RLU value .

 (2) Detection of salmonella bacteria contaminated artificially in processed ham

A 90.52% pork cut ham slice from a commercial company C was purchased and divided to have a weight of 10 g. Salmonella cultures were prepared in the same manner as in Example 4, and then 10 g of processed ham were immersed in the salmonella culture solution for 20 minutes each, and then dried on both sides for 30 minutes in the aseptic band. This was homogenized in the same manner as in the lettuce experiment, and then the number of inoculated Salmonella spp. In XLD medium was measured. 5 ml of the homogenized sample was transferred to a sterilized test tube, incubated with a reporter phage for 2 hours RLU values were measured.

The graph of FIG. 5 (B) was obtained based on the number of Salmonella and the RLU value obtained by the above method. This graph is similar to the graph obtained by experiment with pure salmonella culture (Fig. 4). When the R ² value of the graph is close to 1, the number of salmonella present in the processed ham can be estimated relatively accurately by measuring the RLU value .

Processed ham is known to be one of the foods with complicated composition, and therefore it is known that when using the detection method using PCR or immunoassay, accurate results can not be obtained. However, it has been confirmed that the reporter phage of the present invention specifically infects only salmonella bacteria that are artificially contaminated with processed ham, and accurately detects them.

(3) Detection of salmonella contaminated artificially in milk

After purchasing commercially available S milk, 0.5 mL of the serially diluted Salmonella culture at a concentration of 10 ² to 10 ⁶ CFU / mL was inoculated into 5 mL of milk samples, respectively, and 10 ¹ to 10 ⁵ CFU / mL Of contaminated milk samples with salmonella up to.

The salmonella-contaminated milk samples were stored at 4 ° C for 2 hours and then plated on XLD medium to determine the actual number of contaminated salmonella. 5 mL of each sample was transferred to a sterile test tube and inoculated with a reporter phage for 2 hours After culturing, the RLU value was measured.

The graph of FIG. 5 (C) was obtained based on the number of salmonella bacteria obtained by the above method and the RLU value. When this graph was compared with the graph obtained by the experiment with pure salmonella culture (FIG. 4), the intensity of bioluminescence decreased by about 10 times. However, if the R ² value of the graph is close to 1 and the milk-specific standard curve is obtained, the RLU value is measured and compared with this standard curve to estimate the number of Salmonella bacteria present in the milk relatively accurately It will be possible.

The inhibition of the signal of bioluminescence in turbid liquid foods such as milk is known to be a disadvantage of the lux reporter gene. However, it was confirmed that the graph of the experimental result was linear, and it was confirmed that the reporter phage of the present invention can accurately detect Salmonella even in turbid liquid foods such as milk.

The composition for detecting bacterium including the bacteriophage of the present invention and the agent for detecting bacterium can be used for detection of food infections of Salmonella bacteria because it can distinguish live bacteria from dead bacteria, And overcome the disadvantages of immunoassay.

In addition, the bacteriophage is small in size and easy to store, and the phage of the present invention does not require addition of any additional substance, so that it can be developed and used as a small-sized portable kit.

Korea Microorganism Conservation Center (overseas) KCCM11441P 20130801

Claims (7)

In a bacteriophage comprising int (integrase), oac (O-antigen acetylase), puta bactoprenol-linked glucose translocase (GTPA) gene and cps (major capsid protein) operon,
The int , oac , gtrA genes are removed,
Wherein the luxCDABE operon is inserted after the major capsid protein ( cps ) operon and the lux CDABE operon is transcribed by the cps promoter.
delete The method according to claim 1,
The bacteriophage,
SPC32H :: luxCDABE (KCCM 11441P).
A method for detecting salmonella, comprising detecting salmonella from a food using the bacteriophage of claim 1.
5. The method of claim 4,
In the salmonella detection method,
A method for detecting salmonella, comprising infecting a food infected with Salmonella with the bacteriophage of claim 1 and measuring the bioluminescence.
6. The method of claim 5,
In the salmonella detection method,
Wherein the bioluminescence is measured using a luminometer.
5. The method of claim 4,
The food,
Wherein the salmonella detection method is a dairy product containing meat or milk.

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