In 1952, Dulbecco applied the phage plaque technique to animal virology, thus making virus plaque formation a method for titration and study of many viruses.
1. Principle: After the virus infects the cells, due to the limitation of the solid medium, the released virus can only expand to the periphery from the initially infected cells. After several proliferation cycles, a localized diseased cell area is formed, which is the viral plaque. Theoretically, a plaque is formed by a virus particle that initially infects the cell, so this technique is often used for virus particle enumeration and isolation of virus clones. However, in practice, several virus particles often infect one cell at the same time, which affects the accuracy of titration and the homogeneity of clones. For this reason, the inoculated virus solution should be fully dispersed and diluted.
For cell-binding viruses, such as MDV, monolayer cells are required; for cell-releasing viruses, either cells suspended in solid-phase medium or monolayer cells can be used, but the latter requires a solid medium such as agar to cover the cells. To prevent the released virus from flowing in the liquid medium. The concentration of the solid medium depends on the size of the virus, a medium with a lower concentration is used for large viruses, and a medium with a higher concentration is used for small viruses, so as to control the growth rate of plaques within a suitable range. Small plaques need to be observed with a microscope, and large plaques of 1-10mm can be counted with the naked eye. For the convenience of visual observation, dyes such as neutral red are commonly used. Because the diseased cells do not absorb neutral red, colorless plaques appear in the diseased cell area. The titer of the virus suspension is expressed in plaque forming units per milliliter (PFU/ml). For example, the average plaque of 3 cell flasks is 58, the inoculation volume is 0.2ml, and the dilution of the virus is 2.5×103, then the titer of the virus stock solution is: 58÷0.2×2.5×103=7.25×105 (PFU/ ml)
2. Technical application: Plaque technology can be applied to isolate virus clone (clone clone), virus or serum titration, and can also use plaque morphology and size to study the biological characteristics of virus.
(1) Virus biological purification (Virus biological purification) During the serum neutralization test, the titer of the standard virus strain often decreases, thus affecting the accuracy of the test. This situation is more common in insect coal viruses. This may be due to the mixing of the original virus species, or the existence of many mutant virus particles, or even a large number of defective viruses. At this time, it is necessary to purify the standard strains at hand, and use virus plaque technology to select different purified viruses, also known as “clone strains”. The cloned virus was propagated on sensitive cells, the titer was determined, and the appropriate strain was selected for serum neutralization test.
For more efficient purification, unadsorbed virus on the monolayer must be washed with nutrient solution before overlaying the nutrient agar. At the same time, it is necessary to control the concentration of dilution. The number of plaques in a culture flask should not exceed 10, and the plaques of healthy cells within 10mm of them are picked. The picked virus should be passed on for two or more generations. Neutral red dyes are generally believed to inhibit viral plaque formation and damage host cells due to a “photosensitizing” effect. Therefore, cultures with a neutral red overlay should be grown in the dark.
(2) Plaque Reduction Neutralization Test (Plaque Reduction Neutralization Test) Plaque Reduction Neutralization Test is a highly sensitive method for the detection of serum neutralizing antibodies. as the potency. The test uses quantitative virus (100PFU) mixed with the same amount of serum at different dilutions, inoculated with pre-prepared monolayer cells, then covered with nutrient agar and cultured in a 37 ℃ carbon dioxide incubator, and the number of plaques was counted after a few days. , using Karber method to calculate the plaque neutralization titer of the serum. The principle of operation is roughly the same as that of a traditional serum neutralization assay.
Due to the complicated operation of this test, it is rarely used at present, and it has not been adopted as a diagnostic method accepted by all countries in the world. One of the prescribed diagnostic methods.