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Salmonella bacteriophage CKT1 without transduction of drug-resistant gene and application of salmonella bacteriophage CKT1

A Salmonella phage and drug-resistant gene technology, applied to viruses/phages, medical raw materials derived from viruses/phages, applications, etc., can solve the problems of different strain characteristics and different effects, and achieve high outbreaks and low mutation rates , the effect of protecting health

Pending Publication Date: 2022-07-08
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some phage genomes contain drug resistance genes and genes encoding virulence factors; some phages can transfer the virulence genes and drug resistance genes of host bacteria to other bacteria through phage transduction, resulting in the spread of drug resistance genes and virulence factors. The risk of force genes
At present, the phage strains in domestic phage products have different characteristics and different effects, and there are few studies on the evaluation of whether phage can transduce drug resistance genes and virulence genes
In addition, the phage preparation of pathogenic bacteria must be propagated by the host bacteria, so the virulence and safety of the host bacteria need to be evaluated in depth

Method used

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  • Salmonella bacteriophage CKT1 without transduction of drug-resistant gene and application of salmonella bacteriophage CKT1
  • Salmonella bacteriophage CKT1 without transduction of drug-resistant gene and application of salmonella bacteriophage CKT1
  • Salmonella bacteriophage CKT1 without transduction of drug-resistant gene and application of salmonella bacteriophage CKT1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Isolation and culture of Salmonella virulent phage CKT1

[0036] Take 10 mL of centrifugally filtered sewage collected from the farm, add it to 2-fold concentrated LB liquid, add Salmonella pullorum at a ratio of 1%, shake at 37 °C for 8 to 10 hours, centrifuge at 10,000 rpm / min for 3 minutes, take the supernatant with 0.22 A sterile phage-enriched solution was obtained by filtration through a μm filter. Dilute with SM buffer according to a 10-fold gradient ratio, spot plate 10 μL on a double-layer agar plate containing the host Salmonella pullorum, incubate at 37°C for 6-8 hours, and observe the phage plaques. Pick a single phage plaque in SM buffer, shake and mix well and then double-dilute, take 10 μL of each dilution gradient and spot it on a double-layer agar plate. The bacteriophage CKT1 ( figure 1 A). The enriched phages were stained with negative staining method, and the morphology of the phages was observed under a Hitachi H7650 transmission elect...

Embodiment 2

[0039] Example 2: Determination of biological characteristics of bacteriophage CKT1

[0040] Host profile determination: take 10 μL of phage enrichment solution (10 9 PFU / mL) were spotted on double-layer agar plates of different Salmonella, and incubated at 37°C for 6-8 hours. If transparent plaques appeared, it indicated that the host bacteria could be lysed by phage CKT1.

[0041] Table 1: Lysis ability of bacteriophage CKT1 against different Salmonella and Enterobacteriaceae

[0042]

[0043] Note: ++ means it has cleavage ability, - means no cleavage ability

[0044] The results are shown in Table 1. The results show that the phage CKT1 has the ability to lyse the Salmonella enteritidis, Salmonella pullorum and Salmonella typhi and other group D Salmonella of the Salmonella genus.

[0045] Temperature stability assay: will contain 1 mL of 10 8 The centrifuge tubes of PFU / mL phage dilution solution were incubated at 37°C, 50°C, 60°C, 70°C, and 80°C for 2 h, and then d...

Embodiment 3

[0049] Example 3: Analysis of phage CKT1 transduction ability and determination of anti-phage mutation rate

[0050] 1. Verification of the transduction ability of bacteriophage CKT1:

[0051] 10 to culture to stationary phase 8 CFU / mL Salmonella pullorum SPLH19 (containing the drug resistance gene bla TEM , streptomycin resistance gene aadA), add bacteriophage CKT1 to the final concentration of 10 5 PFU / mL, lysed at 37°C for 6 h, centrifuged and filtered, and the phage titer was determined by the double-layer plate method. Take the above phage preparation solution (10 9 PFU / mL) 1 μL, 5 μL and 20 μL were added to 200 μL overnight Salmonella pullorum SPLH12 (sensitive to ampicillin and streptomycin, without resistance gene bla TEM , streptomycin resistance gene aadA), vortex and mix, and incubate at room temperature for 15 minutes to fully adsorb the phage to the bacteria, then add 1 mL of liquid LB, shake at 37 °C for 1 h, centrifuge at 10,000 rpm to discard the supernatan...

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Abstract

The invention discloses a salmonella bacteriophage CKT1 without transduction of drug-resistant gene and application of the salmonella bacteriophage CKT1, the virulent bacteriophage CKT1 is preserved in China Center for Type Culture Collection on January 21, 2022, and the preservation number of the virulent bacteriophage CKT1 is CCTCC (China Center for Type Culture Collection) 2022110. The separated salmonella bacteriophage CKT1 has splitting activity on D-group salmonella such as salmonella pullorum, salmonella gallinarum and salmonella enteritidis from different sources, is short in incubation period, high in progeny bacteriophage outbreak amount and low in bacteriophage mutation resistance rate, does not carry virulence genes and drug-resistant genes on a genome, does not have transduction capability, and can be used for preparing the salmonella bacteriophage CKT1. The bacteriophage does not cause transmission of drug-resistant genes, is a safe, efficient and high-specificity virulent bacteriophage, and does not destroy intestinal flora of chicks. The bacteriophage preparation can become a novel environment-friendly auxiliary means for purifying salmonella pullorum in a breeding chicken farm, and is also an alternative strategy for preventing and treating salmonella pullorum or salmonella gallinarum infection of chicken flocks.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Salmonella phage CKT1 without the ability to transduce drug resistance genes and its application. Background technique [0002] Salmonella belongs to Enterobacteriaceae and is an important pathogen in food-borne diseases. According to statistics, about 70-80% of food-borne diseases in my country are caused by Salmonella infection. In my country, the main prevalent group D Salmonella include Salmonella enteritidis, which can cause zoonotic infection, and Salmonella pullorum, which can cause high mortality in chickens. It is known that poultry is the main host of Salmonella. Among them, Salmonella pullorum not only spreads horizontally between groups, but also spreads vertically to the next generation, causing extremely high mortality to chicks and seriously reducing growth performance; There are no obvious symptoms, but it can spread widely in animals and the environment, causing h...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04A23K10/18A23K50/75A01N63/40A01P1/00
CPCC12N7/00A61K35/76A61P31/04A23K10/18A23K50/75A01N63/40C12N2795/10321C12N2795/10331C12N2795/10332Y02A50/30
Inventor 孙淑红郝桂娟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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