CN117946979A - Three virulent enterocolitis yersinia phage with specific molecular targets and application thereof - Google Patents
Three virulent enterocolitis yersinia phage with specific molecular targets and application thereof Download PDFInfo
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- CN117946979A CN117946979A CN202410093492.7A CN202410093492A CN117946979A CN 117946979 A CN117946979 A CN 117946979A CN 202410093492 A CN202410093492 A CN 202410093492A CN 117946979 A CN117946979 A CN 117946979A
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Abstract
The invention discloses three virulent enterocolitis yersinia phages with specific molecular targets and application thereof. Yersinia enterocolitica phage vB-YenP-WW 1 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64014-B1.Yersinia enterocolitica phage vB-YenP-WW2 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64015-B1.Yersinia enterocolitica phage vB-YenP-WX 1 was deposited at the Cantonese microorganism strain collection (GDMCC) at 11/13 2023, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64016-B1. The novel virulent enterocolitis yersinia phages WW1, WW2 and WX1 provided by the invention can crack 34 strains in 186 strains of food-borne enterocolitis yersinia, and the cracking rate is 18.3%; meanwhile, the phage has good heat stability, tolerance to acid and alkali, strong environment adaptability, difficult inactivation in the application process and good inhibition effect on target bacteria in milk and pork. In conclusion, phages WW1, WW2 and WX1 are expected to become a bacteriostatic agent for preventing and controlling the pollution of yersinia enterocolitica in foods, human bodies or environments.
Description
Technical field:
The invention relates to the field of food safety prevention and control, in particular to yersinia enterocolitica lytic phage and application thereof.
The background technology is as follows:
yersinia enterocolitica is a food-borne zoonotic pathogen that is widely found in the body of a variety of wild and domestic animals. Animals can carry the bacteria for a long period of time after being infected and are discharged through feces, thereby causing pollution of food, water sources, environment and the like. The meat products may be contaminated during animal slaughter processing, transportation and sales; milk from the dairy animals may also be contaminated with yersinia enterocolitica.
The disease caused by yersinia enterocolitica is called yersinia disease, which is the most commonly reported bacterial food-borne zoonotic disease in the third largest in the european union. The disease can not only cause fever, abdominal pain, bloody diarrhea, frequent vomiting, etc., but also cause respiratory, cardiovascular, skeletal, connective tissue and systemic diseases or other complications, even death. Because of its psychrophilic nature, it stably breeds and produces pathogenic thermostable enterotoxins (Yst) in a low temperature environment at 4 ℃, so the disease is also known as "fridge disease". In recent years, yersinia has been widely prevalent worldwide, with europe being the most severe, and second, the rate of yersinia in new zealand has been relatively high, and this rate has also risen. During 2013 to 2017, the rate of notification of yersinia new zealand was almost doubled, increasing from 10.8 to 19.2 per 10 ten thousand people infected. In such cases reported in new zealand, approximately 75% are caused by food spread, with more than 50% caused by pork spread. China also suffered 2 pandemics in the 80 s of the 20 th century caused by yersinia disease. Enterocolitis yersinia is easy to attack young animals with lower resistance in the young period, and the infection rate of young mice, piglets, infants and the like is high in relevant literature. The survey data show that in the cases of diarrhea in children under the age of 5, as counted in 7304 cases of diarrhea in children under the age of 5, national province 10 in 2010-2015, the average infection rate was 0.59% from 43 cases of sick children's feces isolated to pathogenic yersinia enterocolitica.
Gastrointestinal infections caused by yersinia enterocolitica are usually self-limiting, i.e. self-healed by the immune system of the body, but in individuals with a low immune function, symptoms of sepsis or invasive infections are particularly pronounced, and subsequently metastases are usually associated with liver and spleen, and mortality of these patients can be up to 50% and need immediate treatment with antibacterial agents. The inherent resistance mechanisms of yersinia enterocolitica are complex, and the combination of the abuse of antibiotics in clinical and livestock production makes the pathogenic bacteria insensitive to many antibiotics in clinic, and the antibiotics are gradually losing effect. This phenomenon forces researchers to find new therapeutic strategies to achieve the goal of preventing and treating yersinia disease.
Phage were first found in 1915 to be a class of highly specific "bacterial killers". Phages are largely classified into lytic phages and lysophages, wherein lytic phages replicate by attaching to host cells, injecting phage DNA, DNA replication, assembling proteins, packaging nucleic acids with head and tail proteins, lysing host cells to release new phages; lysophage can be propagated by integrating the DNA into the host genome. The therapy of bacteriophages as antibacterial agents, which is widely used in the 20 th century, was called phage therapy; then, due to the rise of antibiotics, the place of phage therapy is gradually replaced. Although antibiotics are remarkable in terms of antibacterial effect, the advent of multi-drug resistant strains has led scientists to pay renewed attention to phage therapy. Currently, phage applications have been related to a number of fields of agriculture, aquaculture, food safety, and the like. Among them, the use of phages in foods has made considerable progress since the first phage product applicable to foods was approved by the U.S. Food and Drug Administration (FDA) in 2006. Phage are currently used mainly in three areas of the food industry: (1) Phage added during primary production (during growth of animals and plants, before harvest stage) to eliminate the possibility of plant or animal disease; (2) In biological sanitation processes, phage and lytic enzymes are used primarily to prevent the formation of biofilms on surfaces of equipment and facilities used in food production processes. (3) During storage, the phage can extend the shelf life of the product by inhibiting the growth of pathogenic bacteria in the food.
Current phage therapy is limited mainly in several ways:
First, due to the high specificity of phage, its lytic spectrum is relatively narrow, and the ability of a particular phage product to inhibit bacteria is limited; secondly, part of phages have lysogenic capacity, and the lysogenic phages only enter a cracking cycle under the condition of encountering environmental stress, so that the lysogenic phages cannot quickly and effectively kill host bacteria and can cause the horizontal transfer of drug resistance and pathogenic genes; finally, the stability of phage preparations also affects the practical use of phage, and phage titers need to be kept within an effective range during food transportation and sales. There is a current lack of yersinia enterocolitica phages that can address the above deficiencies in use simultaneously and have clear genetic information and physicochemical properties.
Disclosure of Invention
The invention aims to overcome the defects of the technology, thereby providing three novel enterocolitis yersinia phage and application thereof. The bacteriophage provided by the invention has good thermal stability and pH stability, has good bacterial growth and biological film inhibition effects, and can be used as a single bacteriostat or a bacteriophage composition component for inhibiting the growth of Yersinia enterocolitica in food.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The first object of the present invention is to provide three novel virulent phages of yersinia enterocolitica:
Yersinia enterocolitica phage vB-YenP-WW 1 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64014-B1.
Yersinia enterocolitica phage vB-YenP-WW2 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64015-B1.
Yersinia enterocolitica phage vB-YenP-WX 1 was deposited at the Cantonese microorganism strain collection (GDMCC) at 11/13 2023, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64016-B1..
The inventor separates yersinia enterocolitica phage WW1(Yersinia enterocolitica phage vB_YenP_WW1)、WW2(Yersinia enterocolitica phage vB_YenP_WW2)、WX1(Yersinia enterocolitica phage vB_YenP_WX1), from a sewage water sample collected from Guangzhou market in Guangdong province, and the phage of the invention belongs to a short tail phage virulent phage.
Through genome similarity comparison, the phage of the invention has a whole genome similarity of less than 95% with the existing phage, and the phage is proved to belong to novel phage.
The incubation period of the phage WW1 is 20min, and the lysis amount is 37PFU/cell; the WW2 incubation period is 5min, and the cracking amount is 53PFU/cell; the WX1 latency period was 10min and the amount of lysis was 40PFU/cell.
The phages WW1, WW2 and WX1 have good heat stability and pH stability and have a wider host range.
A second object of the present invention is to provide the use of the above-mentioned phages WW1, WW2, WX1 for the preparation of phage preparations inhibiting Yersinia enterocolitica.
A third object of the present invention is to provide a phage preparation for inhibiting Yersinia enterocolitica, which is characterized by comprising the phage WW1, WW2 or WX1 as an active ingredient.
Preferably, the phage preparation is a preparation that inhibits yersinia enterocolitica in humans, foods or the environment.
A fourth object of the present invention is to provide a characteristic molecular target of the phage WW1, the nucleic acid sequence of which is shown in SEQ ID NO. 8.
A fifth object of the present invention is to provide a characteristic molecular target of the phage WW2, the nucleic acid sequence of which is shown in SEQ ID NO. 9.
A sixth object of the present invention is to provide a characteristic molecular target of the phage WX1, the nucleic acid sequence of which is shown in SEQ ID NO. 10.
A seventh object of the present invention is to provide an identification primer for identifying phage WW1, WW2 or WX1, which, when identifying phage WW1, is: GGCCAGATTCGAATTAAATCC and TATAAGGACTCCGAGTATGTT, which identify primers ACTTGGATGCTCGCGGTC and GTGCTGAATCATTCCACCGA when phage WW2 is identified, and TGTCGTTCGGTCAGATGCAA and TTGGCTACGATGCGGGATAC when phage WX1 is identified.
An eighth object of the present invention is to provide a kit for identifying phage, comprising said identification primers.
The ninth object of the present invention is to provide an identification method for identifying the phage, which uses the identification primer to perform PCR amplification on a sample to be tested, and if the corresponding target fragment can be amplified, the corresponding phage is obtained.
Compared with the prior art, the invention has the following advantages:
The novel virulent enterocolitis yersinia phages WW1, WW2 and WX1 provided by the invention can crack 34 strains in 186 strains of food-borne enterocolitis yersinia, and the cracking rate is 18.3%; meanwhile, the phage has good heat stability, tolerance to acid and alkali, strong environment adaptability, difficult inactivation in the application process and good inhibition effect on target bacteria in milk and pork. In conclusion, phages WW1, WW2 and WX1 are expected to become a bacteriostatic agent for preventing and controlling the pollution of yersinia enterocolitica in foods, human bodies or environments.
Yersinia enterocolitica phage vB-YenP-WW 1 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64014-B1.
Yersinia enterocolitica phage vB-YenP-WW2 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64015-B1.
Yersinia enterocolitica phage vB-YenP-WX 1 was deposited at the Cantonese microorganism strain collection (GDMCC) at 11/13 2023, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64016-B1.
Drawings
FIG. 1 is a schematic diagram of a transmission electron microscope of phages WW1, WW2 and WX 1.
FIG. 2 shows the temperature stability of phages WW1, WW2 and WX 1.
FIG. 3 shows the pH stability of phages WW1, WW2 and WX 1.
FIG. 4 is the optimal multiplicity of infection for phages WW1, WW2 and WX 1.
FIG. 5 is a one-step growth curve of phages WW1, WW2 and WX 1.
FIG. 6 is a schematic representation of the inhibition of the growth of Yersinia enterocolitica in LB broth by phages WW1, WW2 and WX 1.
FIG. 7 is a detection electrophoretogram of novel detection targets specific for phages WW1, WW2 and WX 1.
FIG. 8 shows qPCR quantitative detection results of phages WW1, WW2 and WX 1.
FIG. 9 is a schematic representation of the inhibition of growth of Yersinia enterocolitica in milk by phages WW1, WW2 and WX 1.
Detailed Description
The present invention will be further described in detail below with reference to the drawings in the embodiments of the present invention.
Example 1: isolation and culture of phage
1. Activation and cultivation of host bacteria
All yersinia enterocolitica strains used in this study were supplied by the institute of microbiology, the university of guangdong. All strains were stored in 25% (v/v) glycerol and stored in a-80℃freezer. 30 mu L of the seed preservation solution is taken and inoculated into 3mL of Luria-Bertani (LB) medium, and the culture is carried out at 28 ℃ and 200rpm for about 8-10 hours until the logarithmic phase, and then the culture can be used for the subsequent experiments.
2. Isolation and purification of phages
Water samples were collected 2L from food market field sewer, guangzhou, guangdong, respectively. The sample was centrifuged at 5000g for 10 min. The supernatant was filtered with a 0.45 μm mixed cellulose filter membrane by suction through a vacuum pump. MgSO 4 was added to the liquid after the suction filtration to give a final concentration of 50mM, and the mixture was left to stand for 10 minutes, and suction filtration was performed through a 0.22 μm mixed cellulose filter membrane using a vacuum pump. All filters collected were minced and placed into 50mL of eluent (3% beef extract, 3% tween 80,5mm nacl) formulated. Ultrasonic cleaning is carried out on the eluent for 10min by using an ultrasonic cleaner, and bacteriophage on the filter membrane is ultrasonically moved into the eluent. Filter sterilization was then performed using a disposable syringe and a 0.22 μm filter head. The filtrate can be stored at 4deg.C.
Yersinia enterocolitica seed preservation solution 30 μl was taken in 3mL LB, 28 ℃ and cultured overnight at 200 rpm. The following day, 100. Mu.L of the overnight bacterial liquid was aspirated and added to 3mL of fresh LB medium, incubated at 28℃at 200rpm for about 8 hours, and when the growth had reached the logarithmic phase, 100. Mu.L of the bacterial liquid was aspirated and added to a tube containing 1mL of 2 XLB (2 mM CaCl 2), and 1mL of the above solution after ultrasonic filtration was added thereto, and incubated at 28℃at 200 rpm. The liquid was removed and filtered sterilized by a disposable syringe and 0.22 μm filter head. Repeating the culture expanding step for 5 times, and storing the final culture expanding solution at 4 ℃.
Taking 100 mu L of yersinia enterocolitica logarithmic phase bacterial liquid, adding 5mL of 0.4% LB agar, pouring the liquid onto an LB agar bottom plate, dripping 2 mu L of culture expanding liquid after soft agar is solidified, placing the plate in a constant temperature incubator at 28 ℃ for 5-8 hours, and observing whether transparent plaques appear.
Phage were selected from the spotting results for spot picking purification. In order to obtain purer phages, the phage purification protocol was subsequently modified. Taking an LB agar bottom plate, dipping a phage clear liquid by an inoculating loop, and scribing on the bottom plate; 100 mu L of log-phase host bacterial liquid is added into 5mL of 0.4% LB agar, the mixture is poured forward from the streak end, and the mixture is stood until solidification, placed in a 28 ℃ incubator for cultivation for about 5 hours, after plaque grows, a single clear plaque is selected for streaking, and the steps are repeated. After 6 rounds of purification, 20 μl of host bacterial liquid was added to 1mL of LB broth; plaques were picked with an inoculating loop and stirred in 1mL LB broth. Culturing the mixed solution at 28deg.C for more than 8 hr, and filtering with 0.22 μm filter head to obtain purified phage. Phages WW1, WW2 and WX1 were thus obtained.
Example 2: identification of phage
1ML of log phase host bacterial liquid and 0.1mL of phage are taken, added into a conical flask with 50mL of LB (2 mM CaCl 2), and cultured for 5-6h at 28 ℃ and 200 rpm. Taking out the culture solution, 8000g, and centrifuging for 30min. 30mL of the supernatant was filtered through a 0.22 μm filter, 6mL of polyethylene glycol 8000 (60%) and 4mL of sodium chloride solution (5M) were added, and the mixture was gently stirred with an inoculating loop to mix, and then left at 4℃overnight. The next day, the phage concentrate was obtained by taking out and centrifuging 12000g, centrifuging at 4℃for 20min, pouring out the supernatant, and re-suspending with 1mL SM buffer. The phage collected were negatively stained with 2% phosphotungstic acid on a microporous copper mesh, and the morphology was observed by a transmission electron microscope.
As shown in FIG. 1, the heads of phages WW1, WW2 and WX1 are regular hexagons, presumably in a regular icosahedron structure, which all belong to the brachyotis virus podovirus according to the standard of the latest virus classification system of the International Commission for viral classification (ICTV).
Example 3: phage WW1, WW2 and WX1 genomic analysis
Genomic DNA of phages WW1, WW2 and WX1 was extracted for whole genome sequencing and analysis. Phage DNA is extracted by a phenol chloroform method, a phage genome library is constructed, sequencing is carried out by using an Illumina platform, and then sequences are spliced by using SPAdes v.3.13.1 splicing software. Similarity of phages WW1, WW2 and WX1 to the reported phage genome was determined by NCBI BLASTn.
As can be seen from sequencing and analysis, the genome sizes of phages WW1, WW2 and WX1 are 39589bp, 40726bp and 40503bp respectively, wherein the DNA of WW2 and WX1 is linear DNA and the DNA of WW1 is circular DNA. The results of the genomic similarity of phages WW1, WW2 and WX1 to the existing phages are shown in tables 1, 2 and 3. According to BLASTN results, the whole genomes of phages WW1, WW2 and WX1 are less than 95% similar to the whole genomes of known phages, and less than 95% similar to each other, indicating that phages WW1, WW2 and WX1 all belong to the novel phage. The virulence gene and drug resistance gene analysis is carried out on the phage genome, and genes related to virulence or drug resistance are not found, which shows that phages WW1, WW2 and WX1 can be used as safe antibacterial agents for preventing and controlling food-borne enterocolitis yersinia.
Table 1: phage WW1 was aligned with NCBI existing phage genome similarity
Table 2: phage WW2 and NCBI existing phage genome similarity alignment
Table 3: phage WX1 was aligned with NCBI existing phage genome similarity
Phage WW1 was predicted to co-annotate 48 Open Reading Frames (ORFs), 28 of which are known functional protein-encoding genes; phage WW2 was annotated to 54 Open Reading Frames (ORFs), of which 39 ORFs are known functional protein-encoding genes; phage WX1 was annotated to 48 open reading frames, of which 22 ORFs are known functional protein-encoding genes. These ORFs can be divided into structural, cleavage, packaging, DNA metabolism, DNA injection and other domains.
The phage cleavage domain gene function classification is shown in Table 4, and the coding sequence is shown in sequence tables SEQ ID 1-7.
Table 4: phage WW1, WW2 and WX1 lytic domain gene functional classification
Yersinia enterocolitica phage vB-YenP-WW 1 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64014-B1.
Yersinia enterocolitica phage vB-YenP-WW2 was deposited at the Cantonese microbiological strain collection center (GDMCC) at 2023, 11, 13, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64015-B1.
Yersinia enterocolitica phage vB-YenP-WX 1 was deposited at the Cantonese microorganism strain collection (GDMCC) at 11/13 2023, address: building 5, road 100, college 59, guangzhou city martyr, post code: 510070, accession number is: GDMCC No:64016-B1.
Example 4: phage stability assay
For the heat stabilization experiment, the titer of phage is firstly adjusted to be 1X 10 9 pfu/mL, then phage are respectively placed at different temperatures for incubation for 1h, and then the titer is measured; for the pH stability experiments, 200mL of LB broth was taken, its pH was adjusted to 1-13, respectively, 10mL of LB broth corresponding to each pH was aspirated into 15mL conical bottom centrifuge tubes, and then filtered with a sterile 0.22 μm filter head. The titer of phage was adjusted to 1X 10 8 pfu/mL, 100. Mu.L of phage solution was added to 900. Mu.L of LB broth of different pH, mixed well and incubated at 28℃for 1h, followed by determination of the titer.
As shown in fig. 2, 3, in terms of pH, phage WW1 remains stable in potency at ph=5-11, phage WW2 remains stable in potency at ph=4-12, phage WX1 remains stable in potency at ph=5-11, wherein phage WX1 is more tolerant of extreme pH conditions than phage WW1, WW2 (fig. 2); in terms of temperature, phage WW1 remained essentially stable in titer at 4-40℃and died at 70℃and phage WW2 remained essentially stable in titer at 4-60℃and died at 70℃and phage WX1 remained essentially stable in titer at 4-50℃and gradually lost activity at temperatures above 50℃and died at 70℃ (FIG. 3).
Example 5: determination of the optimal multiplicity of infection of phage (multiplicity ofinfection, MOI)
The definition of phage MOI refers to the ratio of phage particle count to host bacteria particle count. The MOI conditions yielding the highest phage titers were defined as the optimal MOI. Phage were diluted to 10 9 to 10 3 pfu/mL according to the measured titers, bacteria cultured to the logarithmic phase were vortexed and mixed well, 200. Mu.L of bacteria liquid was sucked and added into a 96-well plate, OD 600 was measured, colony numbers were calculated according to a standard curve equation, and then colony numbers of host bacteria were diluted to 1X 10 7 cfu/mL with SM buffer. 100. Mu.L of phage of the corresponding titer and 100. Mu.L of 1X 10 7 cfu/mL of host bacteria were added to 3mL of LB (2 mM CaCl 2) at MOI=10 2、101、1、10-1、10-2、10-3、10-4 and incubated overnight at 28℃and 200 rpm. After the completion of the culture, the mixture was filtered with a 0.22 μm filter, 100. Mu.L of phage solution was collected after mixing, and the titer was measured by the double-layer plate method, and the experiment was repeated 3 times.
As shown in fig. 4, phage WW1 has a titer of less than 10 8 pfu/mL under the conditions of moi=10 2、101, 1; the titer of the phage is kept above 10 8 pfu/mL under the condition of MOI=10 -1、10-2、10-3、10-4, wherein the maximum titer is 8.97X10- 8 pfu/mL under the condition of MOI=10 -4, so that the optimal MOI of the phage is=10 -4; phage WW2 has a titer of 10 8 pfu/mL or higher under the condition of moi=10 2、101、1、10-1、10-2、10-3、10-4, wherein the highest titer is 0.77×10 9 pfu/mL under the condition of moi=10 -4, so that the optimum moi=10 -4 of the phage; phage WX1 has a titer higher than 10 8 pfu/mL at all MOI conditions, and highest at moi=10 -3, at 7.98×10 9 pfu/mL, so the optimum MOI for phage WX 1=10 -3. It was found that phages WW1, WW2 and WX1 showed a strong lytic effect against Yersinia enterocolitica at a low titer.
Example 6: phage one-step growth curve
The colony count of the host bacteria was adjusted to 10 9 cfu/mL, and 1mL or more of the bacterial liquid was collected and centrifuged at 12000g for 5min. The supernatant was discarded and the pellet was resuspended in 1mL SMbuffer (2 mM calcium chloride). Phage were added at MOI=10 -1, mixed well, and then left to stand at 28℃for 5min, and centrifuged at 12000g for 5min. Subsequently, the supernatant was aspirated for later use, the pellet was resuspended in 1mL of LB broth, 0.1mL of the broth was added to 9.9mL of LB broth (2 mM calcium chloride), and after mixing 1mL was placed in a 1.5mL centrifuge tube, i.e., 0 point. The broth was then incubated at 28℃and 200rpm, with samples taken every 5min until 90min. For each sampling, the liquid was filtered off with a 0.22 μm filter head and the titer was then determined. Experiments were repeated 3 times. And calculating the cracking quantity according to a formula. Amount of lysis = average plaque number during lysis/average plaque number during latency.
The specific results refer to fig. 5. The incubation period of phage WW1 is 20min, the lysis period is 35min, and the lysis amount is 37PFU/cell; the incubation period of phage WW2 is 5min, the lysis period is 20-25min, and the lysis amount is 53PFU/cell; the incubation period of phage WX1 was 20min, the lysis period was 40min, and the lysis amount was 34PFU/cell. The above data demonstrate that phages WW1, WW2 and WX1 can grow rapidly and destroy host cells rapidly, which is beneficial for phage therapy for protecting foods or various materials from bacterial colonization.
Example 7: phage host profiling
Phage host spectra were determined using the spotting method. 100. Mu.L of the log phase bacterial liquid was added to 5mL of LB containing 0.4% agar, and the mixture was poured onto an LB agar plate. Standing in an ultra-clean workbench for 5min, and adding 2 mu L of phage stock solution onto a soft agar plate after the upper agar solidifies. The plates were then placed in a28℃incubator for stationary culture. The results were observed for about 5 hours. And (3) preliminarily judging whether the experimental bacteria are host bacteria of the phage according to whether plaques of star points are formed or not finally.
To exclude the effect of bacteriocins, the spotting was performed by the EOP method after selection of possible host bacteria. They were first diluted to 10 -4 with LB broth, 100. Mu.L of log phase bacteria were added to 5mL of LB containing 0.4% agar by double plate method, and mixed well and poured onto LB agar plates. Standing for 5min in an ultra-clean workbench, and respectively adding 2 mu L of each diluted gradient phage solution onto an agar plate after the upper agar solidifies. Then the flat plate is placed in a 28 ℃ incubator for static culture, and the result can be observed about 5 hours. If the plaque becomes smaller with the dilution gradient, the bacterium can be proved to be the host bacterium of the phage.
As shown in Table 2, the lysis conditions of phage pair 186 Yersinia enterocolitica were measured by the spotting method, phage WW1 could lyse 3/186 Yersinia enterocolitica, phage WW2 could lyse 18/186 Yersinia enterocolitica, and phage WX1 could lyse 14/186 Yersinia enterocolitica. After three phages were made into cocktails, 34/186 yersinia enterocolitica were total lysed. The specific results are shown in Table 5.
Table 5: determination of phage WW1, WW2 and WX1 host spectra
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Example 8: yersinia experiment of phage control of enterocolitis
To explore phage application prospects, phage control enterocolitis yersinia experiments were performed. Taking a single colony of host bacteria, inoculating the single colony into 3mL of LB broth, and culturing the single colony to a logarithmic phase at 28 ℃ and 200 rpm; bacterial colony count was adjusted to 1X 10 7 cfu/mL with SM buffer and phage titers were adjusted to 10 9 to 10 3 with LB broth. 100. Mu.L of host bacterial liquid and 100. Mu.L of phage liquid are taken and added into 3mL of LB (2 mM CaCl 2) according to the ratio of MOI=10 2、101、1、10-1、10-2、10-3、10-4, after uniform mixing, 200. Mu.L of uniform mixing liquid (6 parallel) is added into each well of a 96-well plate, and the control group is LB liquid medium only added with the same amount of bacterial liquid. After all the materials are added, the materials are put into an enzyme-labeled instrument for measurement, OD 600 is measured every 30min, and the total measurement time is 48 hours. Experiments were repeated three times in total.
As shown in fig. 6, WW1 can completely inhibit the growth of host bacteria within 15 hours and WW2 can completely inhibit the growth of host bacteria within 48 hours under the condition of moi=10 2; WX1 can completely inhibit the growth of host bacteria within 14 h. Namely, phages WW1, WW2 and WX1 have better disinfecting effect on Yersinia enterocolitica, wherein the prevention and control effect is most prominent by phage WW 2.
Example 9: phage-specific molecular target mining and validation
In order to further verify the specificity of phage, according to Blast results of a GeneBank database, DNAMAN software is used for finding out differential fragments of phage genome sequences with higher homology of phage WW1, WW2 and WX1 with other phage genome sequences, and screening is carried out to obtain the phage specificity gene fragments; and designing a specific PCR amplification Primer by using Primer3, and obtaining the phage specificity molecular target after PCR amplification verification, wherein the nucleotide sequence of the gene fragment is shown as SEQ ID NO.8, 9 and 10.
Example 11: establishment of phage WW1, WW2 and WX1 molecular target rapid detection method
Based on the final target sequence, a pair of specific amplification primers were designed, the primer sequences are shown in Table 6.
TABLE 6 primer sequences for PCR identification of molecular targets characteristic of phages WW1, WW2 and WX1
PCR was performed using genomic DNA of other phages isolated from food samples in the laboratory as template.
The PCR reaction system was 12.65. Mu.L, which includes: DREAMTAQ GREEN PCR MASTER Mix (2X) 6.25. Mu.L, 5.5. Mu.L of sterile double distilled water, 0.3. Mu.L of each of the forward and reverse primers, and 0.3. Mu.L of DNA template.
The PCR reaction conditions were: pre-denaturation at 95℃for 10min; denaturation at 95℃for 45s; annealing at 54 ℃ for 30s; extending at 72 ℃ for 1min; a total of 30 cycles; finally, the extension is carried out for 10min at 72 ℃.
Detecting PCR products by agarose gel electrophoresis (agarose concentration is 2.0%), and if the electrophoresis result shows that the amplification products have single amplification bands at the target bands, indicating that the sample contains target phage; if no corresponding single amplified band is present, it is indicated that the sample does not contain the target phage. The results of electrophoresis of phages WW1, WW2 and WX1 are shown in FIG. 7A, B, C, respectively, and the results are shown in tables 7, 8 and 9.
Table 7: specificity test results of phage WW1 molecular target in 21 phages
Table 8: specificity test results of phage WW2 molecular target in 21 phages
Table 9: specificity test results of phage WX1 molecular target in 20 phages
In summary, the molecular tag of the present invention is specific to Yersinia enterocolitica phages WW1, WW2 and WX1, and is not detected in other Yersinia enterocolitica and phages, and therefore, can be used as a specific tag specific to the above phages.
Example 12: quantitative detection method for phage specificity detection target
The present example provides a method for quantitative detection of Yersinia enterocolitica phages WW1, WW2 and WX1 in a sample. After the phage DNA concentration was measured using a Qubit fluorescent quantitative apparatus, the sample DNA to be measured was subjected to PCR amplification on a fluorescent quantitative amplification apparatus using the primers in example 11 by 10-fold gradient dilution with sterilized ultrapure water, and 3 parallel experiments were performed for each template, respectively.
The qPCR detection system was 20. Mu.L, which included: TB Green Premix 10. Mu.L, forward and reverse primers each 1. Mu.L, template DNA 0.5. Mu.L, sterilized double distilled water 7.5. Mu.L.
The qPCR amplification procedure was:
qPCR result reading: by Roche The system is amplified and detected on a 96 fluorescent quantitative amplification instrument, and software/> isutilized96SW 1.1 reads the amplification result. If the fluorescent signal is generated on the premise that the blank control does not exist, the sample contains the yersinia enterocolitica phage corresponding to the detection primer; if no fluorescent signal is generated, the sample does not contain Yersinia enterocolitica phage corresponding to the detection primer.
Drawing a standard curve: taking the logarithm of phage DNA dilution as the abscissa and the real-time Cq value of the corresponding qPCR as the ordinate, the curve obtained by fitting is the standard curve of phage quantitative detection, as shown in figure 9. The yersinia enterocolitica phage WW1 DNA concentration has a linear relation with Cq value between 3.48 multiplied by 10 -4 and 3.48 ng/. Mu.L, and the detection limit is 3.48 multiplied by 10 -4 ng/. Mu.L; the phage WW2 DNA concentration was linearly related to the Cq value between 6.83×10 -7 -6.83 ng/. Mu.L, with a detection limit of 6.83×10 -7 ng/. Mu.L; phage WX1 DNA concentration was linearly dependent on Cq value between 6.88×10 -3 -6.88 ng/. Mu.L with a detection limit of 6.88×10 -3 ng/. Mu.L.
Example 13: bacteriostasis of phages WW1, WW2 and WX1 in milk
A single colony of the host bacterium was picked up in 3mL of LB broth and cultured overnight at 28℃and 200 rpm. The following day, the bacterial concentration was adjusted to 10 6 cfu/mL according to the bacterial standard. 5mL of milk is taken in a 50mL centrifuge tube, and 100 mu L of host bacterial liquid and phage with corresponding concentration are added to make MOI 10 3 and 10 4 respectively. Samples were allowed to stand at 4℃and were sampled at regular intervals, and were subjected to gradient dilution and plate coating counting.
As can be seen from fig. 9, phage WX1 showed excellent inhibitory ability in milk. After 6h of co-incubation, the bacterial numbers in the experimental group to which WX1 was added were significantly reduced by 1.2log 10 cfu/mL and 1.57log 10 cfu/mL, respectively. The inhibition was continued for up to 24 hours, at MOI of 10 3 and 10 4, the colony count in the milk was reduced by 1.71log 10 cfu/mL and 2.03log 10 cfu/mL, respectively. In a word, the bacteriostasis effect of the phage WX1 in milk is obvious, and the phage WX1 has a certain potential for preventing and controlling the pollution of the enterocolitis yersinia in foods.
Compared with WX1, phages WW1 and WW2 have better inhibition effect and have larger application value. As can be seen from fig. 9A, B, phage WW1 can reduce the colony count by 3log 10 CFU/mL at 3h under the condition of moi=10 3, and can reduce the colony count to below the detection limit within 6-24h, while for the moi=10 4 treatment group, all host bacteria in milk can be killed at 3 h; phage WW2 showed similar killing effect to WW1 at moi=10 4, and WW2 reduced host colony count from 5log 10 cfu/mL to 2.39log 10 cfu/mL over 0-12h and bacteria count to an uncountable level at 24h under moi=10 3.
In summary, according to the embodiment of the invention, the enterocolitis yersinia phages WW1, WW2 and WX1 are separated and screened from the Guangzhou market in Guangdong province, have high bactericidal activity on the enterocolitis yersinia, can effectively inhibit the growth of the enterocolitis yersinia, and have good application prospects.
SEQ ID NO.1 (gp 47-WW1 endolysin nucleotide sequence)
ATGAGTAAGGTACAATTCAAACCACGCGCTGTGACAGAAGCAATCTTTGTCCACTGTAGCGCAACCAAAGCGTCCATGAATGTTGGGCTGCGTGAAATCCGTCAGTGGCATAAAGAACAAGGCTGGCTTGATGTAGGCTACCACTTCATTATTCGCCGTGATGGGACAATCGAAGAAGGCCGTCCGGTCGATGTCGTAGGGTCTCACGTTAAGGACTGGAATAGTAAGTCAGTCGGTGTGTGCCTCGTAGGTGGCATTGACGATAAGGGCAAACACGAAGCTAACTTTACGCCAGCACAGATGCACTCTCTTAAAGAGAAACTCGCAGACCTTCTGGACATGTATCCAGATGCTGAAGTGAAAGCTCACCATGACGTGGCACCTAAAGCCTGTCCGTCCTTCAACTTGAGCCGATGGCTGAAGACTGGAGAACTGGTTACAAGCGATTGGGGTTAA
SEQ ID NO.2 (gp 22-WW1 type IIholin nucleotide sequence)
ATGTTGTCATTAGATTTTAATAACGAGGTAGTGAAGGCTGCTCCGATTGTAGGGACAGGTGTAGCCGATGGTGCTGCCCGTCTGTTCTTCGGACTGTCCCTTAACGAGTGGTTCTATGTAGCTGCAATTGCCTACACAGTGGCTCAAATTGGTGCCAAGGTAGTCGATGTGATTATCAAATGGAAGAAGGAGGGTAAAGATGTCTAA
SEQ ID NO.3 (gp 6-WW2 holin nucleotide sequence):
ATGTTATCATTAGACTTCAACAACGAAATCATTAAGGCTGCGCCCATTATTGGCACAGGAGTTGCTGATGGGGCGGCCAGACTCTTTTGGGGTCTGTCATTAAACGAGTGGTTCTACATTGCAGCTATCGCCT ACACAGTGGTTCAGATTGGTGCCAAGGTGGTCGATAAGATGATTGATTGGAAGAAAGCTAATAAGGAGTGA
SEQ ID NO.4 (gp 8-WW2 phase Rz-LIKE LYSIS protein nucleotide sequence):
ATGCTGGAATTTTTACGTAAGCTGGTCCCATGGGTTCTCGCTGGGACGCTATTCGGATGGGGATGGCAACTTGGGGCAGACTCAATGGATGCCAAGTGGAAACAGGAGGTACAGAATGAGTACGTTAAGAGAGTTGAGGCTACAGCGAGCACTCAAAGAGCACTCAATGAAATATCGGCTAAGTATCAAGAAGACCTTGCCGCGCTGGAAGGGAGCACTGATAGGATTATTTCTGATTTGCGTAGCGACAATAAGCGGTTGCGCGTCAGAGTCAAAACTACCGGAACCTCCGATGGTAAGTGTGGATTCGAGCCTGATGGTCGAGCCGAACTTGACGAGCGAGATGCTAAAAGTATTCTCGCAGTGACCCAAAGGGGCGACGCTTGGATTCGTGCTCTACAGGATACCATACGCGAACTACAGCATAAGCAGGAGGTTAAGTAA
SEQ ID NO.5 (gp 29-WW2 endolysin nucleotide sequence):
ATGGCTCGTGTACAGTTTAAACCGCGTGAATCTACTGACGCAATCTTTGTCCACTGCTCGGCTACCAAGCCAAGTCAGAATGTAGGTGTCCGTGAGATTCGCCAGTGGCATAAAGAGCAGGGTTGGCTTGATGTGGGATACCACTACATCATCAAGCGCGATGGCACTGTAGAGGAAGGCCGAGATGAGATGGCTGTAGGTTCTCACGCTAAGGGCCACAATCACAACTCAATCGGTGTCTGCCTTGTAGGTGGTATCGACGATAAAGGTAAGTTCGAAGCTAACTTTACGCCAGCACAAATGCAATCCCTCCGCTCACTGCTTGTCGCACTACTGGCTAAGTATAAAGGCGCTGTTCTTCGAGCGCATCACGATGTGGCTCCGAAGGCTTGCCCTTCGTTCGACCTTAAGCGTTGGTGGGAGACGAACGAACTGGTCACTTCTGACCGTGGCTAA
SEQ ID NO.6 (gp 9-WX1 type II holin nucleotide sequence):
ATGCTGTCTTTAGATTTTAACAATGAGTTAGTCAAGGCTGCGCCTATTGTCGGTACGGGCGTGGCTGATGGCGCTGCGAGGCTTTTCTTCGGGCTGAGTCTTAATGAGTGGTTCTATGTGGCTGCTATCGCCTATACAGTGGTTCAGATTGGTGCCAAGGTAGTCGATAAGATGATTGACTGGAAGAAAGCCAATAAGGAGSEQ ID NO.7(gp9-WX1 endolysin Nucleotide sequence):
ATGACTCGTGTACAGTTTAAACAACGTGAATCTACTGACGCAATTTTTGTTCACTGCTCGGCTACCAAGCCAAGTCAGAATGTAGGTGTCCGTGAGATTCGTCAGTGGCATAAAGAGCAAGGTTGGCTTGATGTCGGGTATCATTTTATCATCAAGCGCGATGGCACTGTAGAGGAAGGTCGCGACGAGATGGCTGTGGGTTCACACGTTAAGGGTCACAACCATAACTCAATCGGTGTCTGCCTTGTTGGTGGTATTGACGATAAAGGTAAGTTCGAAGCTAACTTTACACCAGCCCAAATGAAATCCCTTCGCTCACTGCTTGTCACACTACTGGCTAAGTACGAAGGCGCTGTACTGAAAGCCCATCACGATGTAGCACCCAAGGCTTGCCCTTCGTTCGACCTTAAGCGTTGGTGGGAAACTAATCAATTAGTAACGTCTGATCGTGGCTAA
specific recognition nucleic acid sequence of phage WW1 of SEQ ID No. 8:
TTACATCCACTGCTTCAGGCCAGATTCGAATTAAATCCTGTGTTATAAAACCGGCGCGAGACTTCT TACCTTCGATGTGCACAGTTCGTCCTTTTTTGTCCACATACTCGGAGTCCTTATACTTATAGGACACAGSEQ ID NO.9 Specific recognition nucleic acid sequence of phage WW 2:
GGTCACTTGGATGCTCGCGGTCGTCGAATCGTGAACGTAGCGGATGGTATTGAACCCGGCGATGCAATAAATCTAGGCCAAGTCTCTCGGTGGAATGATTCAGCACTGAACTCAAAGAACGCCGCGAAGGTCTCTCCGAAGAACGTGATCG
SEQ ID NO.10 specific recognition nucleic acid sequence of phage WX 1:
TGTCGTTCGGTCAGATGCAACGATTCGACGGATCTACATACAACTCCATGATTGCCGCTAAGGCATCTGAGACTAACGCTAAGACCTCTGAGATGAACTCAGCGGCGAGTGCACTATCTTCCAAGAACGAAGCTGACCGTGCTAAGACTGAGGCTGACCGCACTAACGGTAAGGCTGATGAGGCCGCTGCGAGCGCTACAATAGCCAACGAGAGCGCCATAACCGCTACACAAGGTGCTAGTACAGCTACCACTAAGGCCGCTGAAGCGAAAGACTATGCGGACCGTCTGAACGACTTTGTGACCATTCAGGACCGTATCAACTCTGTGGCTGTGTCTCAGGTAGGTGATGTAGAGCCACATGTATCCCGCATCGTAGCCAA
Claims (10)
1. A phage of yersinia enterocolitica, characterized by any one of the following:
Yersinia enterocolitica phage vB _ YenP _ww1 with deposit number: GDMCC No:64014-B1;
Yersinia enterocolitica phage vB _ YenP _ww2 with deposit number: GDMCC No:64015-B1.
Yersinia enterocolitica phage vB _ YenP _WX1, accession number is: GDMCC No:64016-B1.
2. Use of the phage of claim 1 for the preparation of a phage preparation that inhibits yersinia enterocolitica.
3. A phage preparation for inhibiting yersinia enterocolitica, characterized by comprising the phage of claim 1 as an active ingredient.
4. The phage preparation of claim 1, wherein the phage preparation is a preparation that inhibits yersinia enterocolitica in humans, foods or the environment.
5. The molecular target characterizing Yersinia enterocolitica phage vB _ YenP _ww1 as defined in claim 1, wherein the nucleic acid sequence is as set forth in SEQ ID No. 8.
6.Yersinia enterocolitica phage vB_YenP_WW2, wherein the nucleic acid sequence is as shown in SEQ ID NO. 9.
7.Yersinia enterocolitica phage vB_YenP_WX1, the nucleic acid sequence of which is shown as SEQ ID NO.
Shown at 10.
8. An identification primer for identifying a phage as claimed in claim 1, wherein when identifying phage WW1, the identification primer is: GGCCAGATTCGAATTAAATCC and TATAAGGACTCCGAGTATGTT, which identify primers ACTTGGATGCTCGCGGTC and GTGCTGAATCATTCCACCGA when phage WW2 is identified, and TGTCGTTCGGTCAGATGCAA and TTGGCTACGATGCGGGATAC when phage WX1 is identified.
9. A kit for identifying a phage according to claim 1, comprising an identification primer according to claim 8.
10. An identification method for identifying a phage as claimed in claim 1, characterized in that the identification primer as claimed in claim 8 is used for identifying a sample to be tested, if the corresponding product can be amplified, the corresponding phage.
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