Brucella phage A001 and its application

Abstract

The present invention relates to the technical field of microorganisms and microorganism detection, and specifically to a strain of Brucella phage A001 (Brucella phage A001) and its application. The deposit number of the Brucella phage A001 is CGMCC No.45382. The phage has a lysis effect on Brucella of bovine, swine, sarin rat and melitensis species, has no virulence factors and drug resistance genes, has safety and effectiveness, and can be used for live bacteria detection in contaminated food and environment. The present invention also provides an application of Brucella phage A001 in Brucella detection, which has short detection time, high sensitivity, strong specificity, and can distinguish live bacteria from dead bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and microorganism detection, and in particular to a Brucella phage A001 (Brucella phage A001) and application thereof. Background Art

[0002] Brucellosis exists worldwide and is an important public health issue. The main routes of infection for animals are through contact with sick animals, ingestion of contaminated water and feed, etc. Brucella can cause diseases such as miscarriage, infertility and reduced milk production in animals, causing huge economic losses to the animal husbandry industry. Humans are usually infected with Brucella through direct contact with infected livestock or eating contaminated meat and dairy products. After the onset of the disease, symptoms such as fever, joint pain and sweating appear. These symptoms are not specific and can easily lead to misdiagnosis and neglect of the control of the disease. Infected animals or contaminated unsterilized milk and meat products are the sources of infection of the disease, so strengthening the monitoring of animals and animal-derived products is the key to controlling human brucellosis. Therefore, developing a convenient, rapid and specific detection method is a key issue that needs to be solved urgently.

[0003] At present, 12 types of Brucella have been isolated and identified, and the dominant epidemic strain in my country is Brucella melitensis. The main methods for detecting brucellosis are serological methods, pathogenic methods and molecular biological methods. Among them, the isolation culture identification method is the gold standard for brucellosis detection, but the operation is complicated, time-consuming and labor-intensive, requiring professional operators to complete it in a biosafety level 2 or 3 laboratory, and there is an infection risk for the operator; the serological method has good specificity, but it is easy to produce cross-reactions; the molecular biological method has high sensitivity and fast detection speed, but it cannot distinguish between nucleic acid amplification of live or dead bacteria and produce positive results.

[0004] Bacteriophage is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes. Because some of them can cause the lysis of host bacteria, they are called bacteriophages. Bacteriophages must parasitize in living bacteria and have strict host specificity. Therefore, bacteriophages have specific advantages in the field of bacterial detection. Summary of the invention

[0005] In view of the problems existing in the background technology, one of the purposes of the present invention is to provide a Brucella phage A001, which has a lysis effect on Brucella bovis, swine, sarin rat and melitensis. The annotation results of VFDB database and CARD database show that the phage has no virulence factors and drug resistance genes, so phage A001 is safe and effective and can be used in food detection and environmental live bacteria detection.

[0006] Another object of the present invention is to provide an application of Brucella phage A001 in food detection. Based on the real-time fluorescence quantitative PCR of the phage, live Brucella can be indirectly detected with a detection limit of 10 0 CFU / mL has the characteristics of strong specificity, high sensitivity, fast detection speed, and the ability to distinguish between dead and live bacteria.

[0007] To achieve the above object, the present invention provides a Brucella phage A001, the deposit number of the Brucella phage A001 is CGMCC No.45382.

[0008] The Brucella phage A001 described in the present invention is isolated from a strain of Brucella bovis culture in Inner Mongolia Autonomous Region, and can form transparent plaques with clear edges on double-layer agar plates. Under a transmission electron microscope, the Brucella phage A001 has an icosahedral solid head with a head diameter of about 61.0 nm and a tail length of about 17.0 nm, belonging to the family of short-tailed phages. The Brucella phage A001 described in the present invention can lyse smooth Brucella but not bacteria of other genera; it can lyse Brucella in a smaller amount and obtain more phages; it enters the lysis period at 30 minutes, and its titer is stable when it is irradiated with ultraviolet light for 30 minutes at a temperature of 40-60°C and a pH of 2-13, and has high stability.

[0009] Furthermore, the present invention provides a culture, the composition of which includes the Brucella phage A001.

[0010] The titer of the Brucella phage A001 of the present invention is very easy to increase after proliferation. In a specific embodiment provided by the present invention, the culture titer of the Brucella phage A001 is greater than 10 10 PFU / mL.

[0011] Since the bacteriophage must parasitize in live bacteria, it can be proved that the live bacteria required for its own replication exist. Therefore, the Brucella phage A001 of the present invention is applied to the detection of Brucella, and it can be accurately judged whether there are Brucella viable bacteria in the sample to be tested. The present invention can detect Brucella living in the sample, which is very important for the diagnosis, treatment and prognosis of food safety and brucellosis.

[0012] Smooth type Brucella generally has stronger toxicity and pathogenicity. The lysis effect of Brucella phage A001 described in the present invention is especially stronger to smooth type Brucella. It is illustrated that Brucella phage A001 provided by the present invention has a relatively larger application range in the identification application of Brucella.

[0013] The bacteriophage A001 of the present invention infects the host bacteria in a short time, has a fast growth rate and a short reproduction cycle, and can shorten the detection time when applied to food detection.

[0014] The bacteriophage A001 described in the present invention has good tolerance to temperature, ultraviolet light and pH, has high stability in actual production, and has a wider range of usage scenarios.

[0015] Furthermore, the present invention provides a detection primer, which includes the sequence shown in SEQ ID No.1 and / or SEQ ID No.2.

[0016] The detection primers of the present invention are designed based on the bifunctional DNA priming enzyme / polymerase of Brucella phage A001. The detection primers of the present invention can be used to realize the real-time fluorescence quantitative PCR detection of the Brucella phage A001.

[0017] Furthermore, the present invention provides a detection kit, which includes the detection primer.

[0018] Preferably, the detection kit further comprises the Brucella phage A001 and / or the culture.

[0019] In a more specific embodiment of the present invention, when the detection kit uses a 20 μl reaction system for real-time fluorescence quantitative PCR, the 20 μl reaction system includes 10 μl of 2×T5Fast qPCR Mix, 0.6 μl of 10 μM forward primer, 0.6 μl of 10 μM reverse primer, 2 μl of template and the remainder of water.

[0020] Furthermore, the present invention provides the use of the Brucella phage A001, or the culture, or the detection primer, or the detection kit in Brucella detection for non-disease diagnosis.

[0021] Preferably, the application comprises the following steps:

[0022] S1, mixing the sample to be tested, the Brucella culture medium and the Brucella phage A001 to obtain a mixed culture solution, and culturing;

[0023] S2. Sampling the mixed culture solution at the 0th hour and the nth hour of the culture to obtain samples T0 and T1. n ; Among them, n≥24;

[0024] S3, respectively, with sample T0 and sample T n The detection primers are used to perform real-time fluorescence quantitative PCR on the amplification substrate to obtain the Ct0 value corresponding to sample T0 and the Ct0 value corresponding to sample T1.n The corresponding Ct n value;

[0025] According to Ct n The difference between the Ct value and the Ct0 value, ΔCt, is used to determine whether the sample to be tested contains Brucella;

[0026] The calculation formula of ΔCt is: ΔCt = Ct n -Ct0;

[0027] Judgment basis: When ΔCt≥1, it is judged that the sample to be tested contains Brucella.

[0028] Preferably, the titer of Brucella phage A001 in sample T0 in step S2 is not less than 10 6 PFU / mL.

[0029] Preferably, the Brucella includes any one or more of the following Brucella: Brucella bovis S19, Brucella suis S1330, Brucella melitensis Isfahan, and Brucella sarcoma 5K33.

[0030] Preferably, the application includes any one of the following:

[0031] (1) Detect whether food contains Brucella;

[0032] (2) Check whether the working environment contains Brucella.

[0033] In addition, described brucella phage A001, or described culture, or described detection primer, or described detection kit also can be used for the brucella detection of disease diagnosis purpose.Any transmission factor that is contaminated by brucella, such as sick animal abortion product, sick animal's milk meat and internal organs, fur, sewage, soil, excrement and dust etc., can detect.

[0034] The real-time fluorescence quantitative PCR method based on bacteriophage A001 of the present invention does not require the extraction of nucleic acid, and the detection time is greatly shortened compared with the traditional culture method. 0 CFU / mL of Brucella, with high sensitivity and strong specificity.

[0035] Beneficial effects:

[0036] The present invention provides a Brucella phage A001, the deposit number of which is CGMCC No. 45382. The Brucella phage A001 has host specificity to Brucella, can lyse smooth bovine, swine and sarin rat Brucella strains at a conventional test concentration (RTD), and can effectively inhibit the growth of Brucella at 10 4Smooth-type Brucella melitensis can be lysed under RTD. It was identified as Brachyurae family of Caudata order by transmission electron microscopy and whole genome sequencing. It has no virulence factors and drug-resistant genes, is safe and effective, and can be used for food testing and environmental live bacteria detection. The optimal infection multiplicity of the Brucella phage A001 is 0.1, the incubation period is 30 minutes, the lysis period is 40 minutes, and it enters the plateau phase after 70 minutes. The lysis amount is 2210PFU / cell. The phage titer of the Brucella phage A001 is stable when the temperature is 40-60°C, the pH is 2-13, and the ultraviolet irradiation is 30 minutes, and it has good thermal, ultraviolet and acid-base stability.

[0037] The present invention also provides an application of Brucella phage A001 in Brucella detection. The Brucella phage A001 provided by the method is used to detect Brucella in food using a fluorescent quantitative PCR method, which can achieve a good detection effect with a detection limit of 10 0 CFU / mL. Compared with traditional culture-based technologies, the Brucella detection method provided by the present invention has higher specificity and sensitivity, shorter detection time, no need to extract nucleic acid, and can distinguish between live and dead bacteria, which helps to provide experimental methods and theoretical basis for rapid detection of Brucella contamination in food.

[0038] Description of biological deposit:

[0039] Brucella phage A001 (Brucella phage A001) was deposited on December 30, 2022 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Brucella phage (Brucellaphage), and the deposit number is: CGMCC No.45382. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0041] Figure 1 is a plaque morphology diagram of bacteriophage A001 described in Example 1 of the present invention;

[0042] Figure 2 is an electron microscope morphology image of bacteriophage A001 described in Example 1 of the present invention;

[0043] Figure 3 This is the result of determining the optimal multiplicity of infection of bacteriophage A001 and Tb described in Example 2 of the present invention;

[0044] Figure 4 is a one-step growth curve diagram of bacteriophage A001 and Tb described in Example 2 of the present invention;

[0045] Figure 5 The results of the temperature sensitivity test of bacteriophage A001 and Tb described in Example 2 of the present invention are as follows;

[0046] Figure 6 The results of the sensitivity test of bacteriophage A001 and Tb to ultraviolet irradiation described in Example 2 of the present invention are as follows;

[0047] Figure 7 The results of the pH sensitivity test of bacteriophage A001 and Tb described in Example 2 of the present invention are as follows;

[0048] Figure 8 is a standard curve diagram of the real-time fluorescence quantitative PCR detection of bacteriophage A001 described in Example 4 of the present invention;

[0049] Fig. 9 It is an amplification curve graph and a melting curve graph of the real-time fluorescence quantitative PCR detection described in Example 4 of the present invention, wherein Figure A represents the amplification curve of the Brucella phage and the negative control strain fluorescence quantitative PCR; Figure B represents the melting curve of the Brucella phage and the negative control strain fluorescence quantitative PCR; in Figure A and Figure B, red represents Brucella phage; blue represents negative control; green represents blank control. DETAILED DESCRIPTION

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.

[0051] Example 1 Morphological characteristics of bacteriophage A001

[0052] 1. Purification and proliferation of bacteriophage A001:

[0053] The double-layer agar method is used to proliferate phages. Plaques of uniform size and shape are selected on the plate. The plaques are placed in broth and soaked overnight. The supernatant is centrifuged and then spread on a double-layer plate after gradient dilution. The above steps are repeated for more than three times of continuous purification. The plaques are uniform in size and shape, and the titer is determined.

[0054] The results are as follows Figure 1 As shown: The plaques of A001 are clear and bright, without halos, with a diameter of 4-6 mm and a titer greater than 10 10 PFU / mL.

[0055] 2. Electron microscopic observation of bacteriophage A001:

[0056] The phage suspension was dropped onto the grid and adsorbed for 5 minutes, and the excess suspension was removed with filter paper. Then, phosphotungstic acid dye was added and stained for 1 minute, and the excess dye was removed with filter paper. After drying at room temperature, the phage morphology was observed under a transmission electron microscope.

[0057] The results are as follows Figure 2 As shown: The purified phage was identified by transmission electron microscopy and its morphology was an icosahedral solid head with a head diameter of about 61.0 nm and a tail length of about 17.0 nm. It belongs to the order Caudovirales, family Brachyuraviridae.

[0058] Example 2 Biological characteristics of bacteriophage A001

[0059] 1. Determination of lysis spectrum of bacteriophage A001

[0060] Brucella bovis S19, 45 / 20, Brucella suis S1330, Brucella melitensis Isfahan, B115, Brucella canis RM6 / 66, Brucella sarcoma 5K33, Escherichia coli and Staphylococcus aureus were selected for lysis spectrum analysis.

[0061] Culture the above strains to the logarithmic phase, take 10 ml of semi-solid culture medium, add 100 μL of bacterial solution, mix well and spread on solid culture medium, after the upper culture medium solidifies, add 8 μL of phage suspension, culture at 37°C for 24-48 hours, and observe the results.

[0062] The results are shown in Table 1: The lysis spectrum of phage A001 was similar to that of reference phage Tb. It had lysis effects on smooth-type bovine, suis and sarin rat Brucella at routine test dilution (RTD); 4 ×RTD), it can cause the lysis of smooth-type Brucella melitensis. However, bacteriophage A001 has a stronger lysis ability against Brucella suis.

[0063] Table 1 Lysis spectra of bacteriophage A001 and Tb

[0064]

[0065] Note: In the table, (S) represents smooth type; (R) represents rough type; + represents lysis; - represents no lysis; ± represents partial lysis.

[0066] 2. Determination of the optimal infection multiplicity of bacteriophage A001

[0067] The concentration of phage suspension was 10 8 , 10 7 , 10 6 , 10 5 , 10 4PFU / mL, the host bacteria concentration was fixed at 10 7 CFU / mL, add phage suspension and host bacterial solution in the ratio of 10, 1, 0.1, 0.01, and 0.001, mix well, and culture in an air shaker at 37°C and 200rpm for 24h. After the culture time is over, filter with a 0.22μm microporous filter to obtain phage suspension, dilute each group of phage suspension, take 100μL of host bacterial solution and 100μL of phage dilutions of different dilutions in each group, mix them thoroughly, and use the double-layer agar plate method to determine the titer. The ratio corresponding to the highest titer is the optimal infection multiplicity of phage infection of the host bacteria.

[0068] The results are as follows Figure 3 As shown in the figure: When the infection multiplicity is 0.1, the titer of A001 reaches the highest, indicating that when the ratio of phage to host bacteria is 0.1, more phages can be proliferated to obtain the maximum benefit. The optimal infection multiplicity of the reference phage Tb is 10, indicating that it requires more phages to lyse Brucella, and its application value is low.

[0069] 3. Determination of one-step growth curve of bacteriophage A001

[0070] The bacteria cultured for 24-48 hours were adjusted to a turbidity of 10 using a turbidity meter. 9 CFU / mL, diluted to 10 7 CFU / mL. Mix equal volumes of phage and host bacteria according to the optimal infection multiplicity, place at 37℃ for adsorption for 10 minutes, centrifuge at 4℃, 5500rpm for 15 minutes, discard the supernatant, and resuspend the bacteria in 10mL liquid culture medium; culture at 37℃, 200rpm, start timing, take liquid culture medium every 10 minutes, until 240 minutes, centrifuge and measure the phage titer in the supernatant liquid culture medium. Draw a one-step growth curve with time as the horizontal axis and the measured titer as the vertical axis. Lysis amount = the titer of the phage at the end of lysis divided by the concentration of host bacteria at the beginning of infection.

[0071] The results are as follows Figure 4 As shown in the figure: the incubation period of bacteriophage A001 is 30 min, which is shorter than that of reference phage Tb. It can enter the lysis period earlier, with a lysis period of 40 min and a lysis amount of 2210 PFU / cell (lysis amount = titer of phage at the end of lysis / host bacteria concentration at the beginning of infection = 2.21×10 10 / 10 7 =2210PFU / cell).

[0072] 4. Determination of the temperature sensitivity of bacteriophage A001

[0073] The phage suspension was divided into Eppendorf tubes and placed in a constant temperature water bath at 40℃, 50℃, 60℃, 70℃, and 80℃. Samples were taken after 2 hours and the phage titer was determined by the double-layer agar method. Three parallels were set for each temperature and the results were averaged. A change curve was drawn with the temperature action time as the horizontal axis and the phage titer as the vertical axis.

[0074] The results are as follows Figure 5 As shown: A001's activity remains basically unchanged at 40-60°C, and when the temperature reaches 70°C, the phage activity gradually decreases, and it is completely inactivated at 80°C. The reference phage Tb's activity gradually decreases at 40-70°C, and it is completely inactivated at 70°C. Therefore, A001 is more tolerant to temperature than Tb.

[0075] 5. Determination of the sensitivity of bacteriophage A001 to ultraviolet light

[0076] Prepare a blank plate, spread the phage solution on the plate, and place it in the center of the biosafety cabinet. Turn on the UV light for irradiation, take samples every 3 minutes from 0 to 30 minutes, and use the double-layer agar method to detect the phage titer.

[0077] The results are as follows Figure 6 As shown: the activity of phage A001 and reference phage Tb gradually decreased after ultraviolet irradiation, but still maintained a high activity after 30 minutes of irradiation.

[0078] 6. Determination of pH sensitivity of bacteriophage A001

[0079] Adjust the pH of PBS with NaOH and hydrochloric acid solution to 1-14. Take equal volumes of PBS liquid culture medium and phage solution with different pH values, mix them well and place them in a constant temperature incubator at 37°C for 2 hours. After the incubation, test the phage titer.

[0080] The results are as follows Figure 7 As shown, both phage A001 and reference phage Tb maintained high activity in the pH range of 2-13, and completely lost their activity at pH = 1 and 14.

[0081] Example 3 Whole genome analysis of bacteriophage A001

[0082] 1. General characteristics of the genome

[0083] The bacteriophage A001 genome was extracted, and the DNA concentration was measured by Nanodrop1000, followed by whole genome sequencing.

[0084] The whole genome sequencing showed that the genome was double-stranded DNA with a total length of 38380 bp and a GC content of 48.17%. BLAST comparison in the NCBI database showed that the coverage rate with BK2 (HF569088.1) was 100% and the consistency was 99.96%.

[0085] 2. Phage gene function prediction

[0086] Gene prediction was performed on bacteriophage A001, and the protein sequence was compared using NCBI blastp to obtain the corresponding annotation information. The results are shown in Table 2.

[0087] Table 2 Gene prediction of bacteriophage A001

[0088]

[0089]

[0090]

[0091] Example 4 Real-time fluorescence quantitative PCR detection of bacteriophage A001

[0092] 1. A method for detecting Brucella in food using Brucella phage A001 and real-time fluorescence quantitative PCR principle:

[0093] The bifunctional DNA priming enzyme / polymerase of Brucella phage A001 was selected to design primers, and the primer pair for real-time fluorescence quantitative PCR was:

[0094] Forward primer: 5'-TTAATCCACCGCGTCACCTC-3' (SEQ ID No. 1);

[0095] Reverse primer: 5'-ATCGTTCGAGGAAGTCGAGC-3' (SEQ ID No. 2).

[0096] Real-time fluorescence quantitative PCR reaction system:

[0097]

[0098] The Brucella phage A001 suspension was mixed with the sample to be tested in a ratio of 1:1, and broth culture medium was added to make up to 3 mL. After mixing, sampling was performed, 10% chloroform was added, and centrifugation was performed at 12000 rpm for 2 min, and real-time fluorescence quantitative PCR was performed. The above mixture was then placed in an air shaker at 37°C for incubation, and samples were taken at intervals (24h, 48h and 72h). The real-time fluorescence quantitative PCR operation after sampling was the same as above.

[0099] When the Ct value of real-time fluorescence quantitative PCR sampled at a certain time point is less than the Ct value of real-time fluorescence quantitative PCR sampled at 0h of culture, it indicates that the phage at that time point has been amplified and the sample may contain Brucella. The difference ΔCt of the two product Ct values ​​is used to qualitatively determine whether there is live Brucella in the sample. The judgment basis is: when ΔCt ≥ 1, it is determined that Brucella is detected, otherwise there is no Brucella in the sample.

[0100] 2. Determination of phage titer

[0101] The phage titer was determined by measuring the optimal infection multiplicity. When the MOI was 0.1, the phage titer reached the maximum. This indicated that the host bacteria concentration was 10 7 CFU / mL, phage titer is 10 6 PFU / mL, the number of progeny phages that proliferate is greater, so 10 6 PFU / mL was used as the infection titer.

[0102] 3. Standard curve and minimum detection limit of qPCR method

[0103] The DNA of A001 was serially diluted 10-fold (10 7 -10 0 copies / 2μl), qPCR detection was performed, and the positive fluorescence signal was checked by melting curve analysis. The logarithm of the gene copy number was used as the horizontal axis and the Ct value was used as the vertical axis to draw a standard curve.

[0104] The results are as follows Figure 8 As shown, this method is 1.00×10 7 -1.00×10 0 The linear relationship is good within the range of copies / 2μl, and the standard curve is y=-3.3398x+33.994, R 2 =0.9954, the amplification efficiency is 99%, and the minimum detection limit is 1.00×10 0 copies / 2μl.

[0105] 4. qPCR method stability test

[0106] In order to evaluate the repeatability and stability of qPCR detection, intra-group and extra-group repeatability experiments were performed on DNA samples, and the coefficient of variation was calculated to evaluate the repeatability and stability of this method.

[0107] The results are shown in Table 3: 7 -1.00×10 0Eight DNA samples with different copy numbers were used as templates for amplification. The standard deviation of the average Ct value within the group was 0.03-0.73, and the coefficient of variation was 0.14%-2.22%; the standard deviation of the average Ct value between groups was 0.23-0.92, and the coefficient of variation was 0.72%-3.65%. The results had a small degree of dispersion, indicating that this method has good repeatability and stability.

[0108] Table 3 Real-time fluorescence quantitative (qPCR) detection stability

[0109]

[0110] 5. qPCR method specificity test

[0111] Specificity testing was performed using 8 Brucella phages (Brucella phage A001, Brucella phage A002, Brucella phage M001, from the Inner Mongolia Autonomous Region Institute of Endemic Disease Control; Iz, Bk2, Tb, R / O, R, from the UK Weiqiao Laboratory) and 11 bacterial strains (Brucella S19, Escherichia coli VP BNCC, pathogenic enterocolitica Yersinia O:9, Vibrio cholerae VC 0139M045, Vibrio cholerae 01VC M6961, Salmonella Agona, Salmonella Typhimurium, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Delpy, Staphylococcus aureus DC50280, available from China Medical Bacteria Collection or China Veterinary Microbiology Collection).

[0112] The results are as follows Fig. 9 As shown: All Brucella phages can be detected using this method, and the blank control test is negative. There are non-specific amplification signals in the negative control samples, which can be identified by melting curve analysis. The melting temperature of positive samples is 84-85℃, and the false positive samples are <80℃.

[0113] 6. Brucella detection limit determination

[0114] Brucella S19 was diluted 10 times in series (10 7 -10 0 CFU / mL), respectively 6 PFU / mL phage was mixed and cultured in an air shaker at 37°C and 200 rpm. Samples were collected after 0, 24, 48, and 72 h of culture, treated with 10% chloroform, and centrifuged at 4°C and 12,000 rpm for 2 min. The supernatant was used for qPCR detection, and the negative control was treated with 10% chloroform. 7 CFU / mL Brucella bacterial liquid, and the blank control used liquid culture medium instead of bacterial liquid.

[0115] The results are shown in Table 4: In different concentrations of Brucella S19, after 24 hours of culture, the bacterial concentration was 10 7 -10 4 The increase of phage titer was detected in the samples with CFU / mL, and the ΔCt range was 2-23 compared with 0h. After 48h of culture, at low concentration (10 3 -10 0 The amplification of phages was detected in the Brucella samples with a concentration of 10 CFU / mL. 0 The titer of phage in the Brucella sample (CFU / mL) was of the same order of magnitude as that in the high concentration. Therefore, the sensitivity of qPCR detection in this method was about 10 0 CFU / mL.

[0116] Table 4 Detection range of qPCR method

[0117]

[0118] 7. Propagation of Brucella phage A001 in spiked milk

[0119] Phage A001 (final concentration 10 6 PFU / mL) and 10 4 CFU / mL Brucella S19, cultured at 37°C in an air shaker at 200 rpm, and samples were collected after 0, 6, 11, 22, 32, 48, and 72 h for qPCR detection and titer determination.

[0120] The results are shown in Table 5: The Brucella content in spiked milk is 10 4 CFU / mL, the phage can remain active and continue to proliferate for 72 h in spiked milk, and qPCR can quickly and effectively detect the amplification of phage within 32 h, while the double-layer agar method can detect phage proliferation at 48 h. This method is more sensitive and accurate than the double-layer agar method.

[0121] Table 5 Detection of bacteriophage proliferation in spiked milk by qPCR and double-layer agar method

[0122]

[0123] 8. Application of qPCR method in detecting Brucella in milk samples

[0124] The titer is 10 6 The phages with PFU / mL were added to milk and mixed with different bacterial solutions (Brucella, Escherichia coli, and Staphylococcus aureus), and cultured in a shaker at 37°C. Samples were taken at 0h-72h for qPCR detection and analysis, and the bacterial solution of the blank control group was replaced with liquid culture medium.

[0125] The results are shown in Table 6: The present invention can detect 10 0 CFU / mL of bovine Brucella, the detection time for higher concentrations of bacteria is relatively shorter (24 hours).

[0126] Table 6 Specificity of qPCR assay in milk samples

[0127]

[0128]

[0129] The above-mentioned embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of the invention patent protection. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Brucella phage ( Brucella phage )A001, characterized in that The deposit number of the Brucella phage A001 is CGMCC No.45382.

2. A culture, characterized in that The composition of the culture includes the Brucella phage A001 according to claim 1.

3. A detection primer, characterized in that: The forward primer of the detection primer is shown as SEQ ID No.1, and the reverse primer of the detection primer is shown as SEQ ID No.

2.

4. A detection kit, characterized in that: The detection kit comprises the detection primers described in claim 3; and also comprises the Brucella phage A001 described in claim 1.

5. Use of the Brucella phage A001 described in claim 1, or the culture described in claim 2, or the detection primer described in claim 3, or the detection kit described in claim 4 in the detection of smooth Brucella for non-disease diagnosis.

6. The use according to claim 5, characterized in that: The steps include: S1, mixing the sample to be tested, the Brucella culture medium and the Brucella phage A001 to obtain a mixed culture solution, and culturing; S2. Sampling the mixed culture solution at the 0th hour and the nth hour of the culture to obtain samples T0 and T1. n ; Among them, n≥24; S3, respectively, with sample T0 and sample T n The detection primers are used to perform real-time fluorescence quantitative PCR on the amplification substrate to obtain the Ct0 value corresponding to sample T0 and the Ct0 value corresponding to sample T1. n The corresponding Ct n value; According to Ct n The difference between the Ct0 value and the Ct0 value is ΔCt, which is used to determine whether the sample to be tested contains smooth Brucella; The calculation formula of ΔCt is: ΔCt=Ct n -Ct0; Judgment basis: When ΔCt≥1, it is judged that the sample to be tested contains smooth Brucella.

7. The use according to claim 6, characterized in that: Step S2: The titer of Brucella phage A001 in sample T0 is not less than 10 6 PFU / mL.

8. The use according to any one of claims 5 to 7, characterized in that: The smooth Brucella includes any one or more of the following smooth Brucella: Brucella bovis S19, Brucella suis S1330, Brucella melitensis Isfahan, and Brucella sarcoma 5K33.

9. The use according to any one of claims 5 to 7, characterized in that: The application includes any of the following: (1) Detect whether food contains smooth Brucella; (2) Check whether the working environment contains smooth Brucella.

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