Method for high-throughput screening of phage host spectrums

A phage and high-throughput technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/testing, can solve the problems of large waste of related reagents and phage, large amount of phage consumption, large amount of culture medium, and the need Long time and other problems, to save time and labor costs, reliable records, short time effect

Inactive Publication Date: 2020-03-10
云南省地方病防治所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the determination methods of phage host spectrum at home and abroad are concentrated on direct drop method and double-layer plate method. Whether it is the traditional direct drop method or the classic double-layer plate method, it takes a long time, and the relevant reagents and The waste of phage is large; about 26ml LB medium is needed for the double-layer plate method, and only

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A method for high-throughput screening of phage host spectrum, specifically comprising the following steps:

[0022] (1) 1 strain of host bacteria (EV76), 92 strains of wild plague phage, preparation of microculture plates, LB liquid medium, and LB semi-solid medium.

[0023] (2) After the wild plague phage preserved at 4°C was rejuvenated at 28°C, 220rpm, and 24h, the titer was measured to make the titer reach 10 6 The above spare.

[0024] (3) The host bacterium (EV76) was shaken in an air bath at a suitable temperature (28°C) until it was ready for use in the logarithmic growth phase, and the background of the host bacterium was clear.

[0025] (4) Add 200ul LB semi-solid medium to the 96-well microculture plate, wait for it to solidify, add 10ulEV76 bacteria solution respectively; leave 2 wells as negative control, add 10ul LB liquid medium, leave 2 wells as positive control.

[0026] (5) In addition to the negative wells, add 5ul plague diagnostic phages to the 2...

Embodiment 2

[0029] A method for high-throughput screening of phage host spectrum, specifically comprising the following steps:

[0030] (1) Host bacteria: 92 strains of Yersinia pestis, related bacteria of Yersinia pestis (Yersinia enterocolitica, Yersinia pseudotuberculosis I-VI, Shigella, Salmonella, Escherichia coli, etc.) Preliminary bacteria (Staphylococcus, Streptococcus, Proteus, etc.), other bacteria (fungi), etc., 1 strain of wild plague phage, preparation of microculture plates, LB liquid medium, and LB semi-solid medium.

[0031] (2) After a wild plague phage strain stored at 4°C was rejuvenated at 28°C, 220rpm, and 24h, the titer was measured to make the titer reach 10 6 The above spare.

[0032] (3) 92 kinds of host bacteria were shaken in an air bath at an appropriate temperature (30°C) until the logarithmic growth phase was ready for use, and the background of the host bacteria was clear.

[0033] (4) Add 200ul LB semi-solid medium to the 96-well microculture plate, wait ...

Embodiment 3

[0037] A method for high-throughput screening of phage host spectrum, specifically comprising the following steps:

[0038] (1) Host bacteria 92 strains of Staphylococcus aureus (ATCC25923), related bacteria of Staphylococcus aureus (Staphylococcus epidermidis, Staphylococcus saprophyticus, etc.), non-related bacteria of Staphylococcus aureus (Yersinia enterocolitica, Yersinia pseudotuberculosis Ⅰ~Ⅵ﹑Shigella ﹑Salmonella ﹑Escherichia coli, etc.), other bacteria (fungi), etc., 1 strain of Staphylococcus aureus phage isolated from sewage, microplate, LB liquid medium, Preparation of LB semi-solid medium.

[0039] (2) Measure the potency of a Staphylococcus aureus phage strain stored at 4°C after rejuvenation at 37°C, 220 rpm, and 24 hours, so that the titer reaches 10 6 The above spare.

[0040] (3) 92 kinds of host bacteria were shaken in an air bath at an appropriate temperature (30°C) until the logarithmic growth phase was ready for use, and the background of the host bacter...

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PUM

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Abstract

The invention discloses a method for high-throughput screening of phage host spectrums and belongs to the technical field of phage host spectrum screening. According to the method disclosed by the invention, the purified bacteriophages are rejuvenated, so that the titer reaches 10<6> or above, air bath shaking culture on host bacteria is carried out at a proper temperature to a logarithmic phase for later use, 100-200 microliters of a LB semi-solid culture medium is added into holes of a 96-hole micro-culture plate respectively, 5-20 microliters of a host bacterium liquid is added after the LBsemi-solid culture medium is solidified, two holes are reserved as negative control, and 5-20 microliters of an LB liquid culture medium is used, 5-20 microliters of diagnostic bacteriophages are added into twp positive control holes, the rest bacteriophages to be detected are respectively added, and after gentle and uniform mixing, the mixture is placed at a proper temperature (18-37 DEG C) for12-24 hours and then is observed. The method provided by the invention can be used for identifying more than 90 bacteriophages or 90 host bacteria each time, the time is short, and the dosage of related reagents and bacteriophages is less.

Description

technical field [0001] The invention relates to a method for high-throughput screening of phage host spectrum, belonging to the technical field of phage main spectrum screening. Background technique [0002] Phage (bacteriophage, phage) is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes. Some of them can cause lysis of host bacteria, so they are called phages. Phages share some of the properties of viruses: they are tiny. The phage genome contains many genes, but all known phages use the ribosomes of bacteria, various factors required for protein synthesis, various amino acids and energy production systems in bacterial cells to achieve their own growth and proliferation. Once outside the host cell, the phage can neither grow nor replicate. Bacteriophages are widely distributed, and wherever there are bacteria, corresponding phages may exist. In human and animal excrement or polluted well water and river w...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12R1/92
CPCC12Q1/04
Inventor 钟佑宏王鹏杨丽华石丽媛董珊珊张海鹏谭红丽
Owner 云南省地方病防治所
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