Preliminary Study On Inactivation Effectiveness And Mechanism Of O-Phthalaldehyde To Bacteriophage MS2

ObjectiveTo provide the further theoretical and experimental data for the bacteriophage MS2 in substitution of poliovirus type I (vaccine strain, PV-â… ) as indicator virus in evaluation of disinfection efficacy, observe the inactivation effectiveness and mechanism of O-phthalaldehyde (OPA) to bacteriophage MS2 in laboratory.MethodsThere are two parts in this dissertation.FIRST PART: Study on inactivation effectiveness of O-phthalaldehyde to bacteriophage MS2 and some biology properties of bacteriophage MS2.1. Subculture bateriophage MS2 and observe resistance to O-phthalaldehyde (OPA) of the 0, 5, 10, 15generation MS2. Analyse the result and compare wheather have differernce.2. Observe stability of bateriophage MS2 under 4℃, 22℃,-20℃and-70℃in 10%glycerol and PBS to explore the best preservation methods of bateriophage MS2.3. Observe the decrease of bacteriophage MS2 under 4℃and room tempreture in different natural water surroundings (hospital waste water, tap water, ultrapure water, lake water.etc.) by mixing certain amount of bacteriophage MS2 solution into the different natural water surroundings. Bacteriophage MS2 were detected and enumerated by the double-agar-layer plaque technique.4. Experiments have been conducted by referring to Technical Standard For Disinfection (2002 edition, Ministry of Health, P.R.China). Study on the inactivation efficacy of O-phthalaldehyde (OPA) to bacteriophage MS2 by suspension tests.SECOND PART: Preliminarily study on the inactivation mechanism of O-phthalaldehyde (OPA) to bacteriophage MS2.1. Observe influence on structure, adsorbtion property and antigenicity of bacteriophage MS2 after inactivation of O-phthalaldehyde (OPA) by 1 %PTA negative staining and transmission electron microscope (TEM) method and serum neutralization test.2. Design specific reverse transcription primer and specific primer of four genes of bateriophage MS2 with prime premier 5.0 software and the GenBank NC₀01417 (the MS2 whole gene sequence) as template. Extract RNA, carry RT-PCR and analyse the results to observe the influence on four genes of bacteriophage MS2 after inactivation of O-phthalaldehyde (OPA).ResultsFIRST PART: Study on inactivation effectiveness of O-phthalaldehyde to bacteriophage MS2 and some biology properties of bacteriophage MS2.1. Bacteriophage MS2 of 0, 5, 10, 15generation show no difference in the resistance to O-phthalaldehyde(OPA) and genetic stability of resistance to O-phthalaldehyde(OPA) retain well.2. There are no obvious difference between stability of bateriophage MS2 under 4℃, 22℃,-20℃and-70℃in 10%glycerol and PBS and the log₁₀ die out values less than 1. Bateriophage MS2 is stable in 10%glycerol, PBS, ultrapure water and nutrient broth. In same preservation solution, stability of bateriophage MS2 under 4°C is better than room temperature,-20℃and-70℃. Preserving under 4℃is a good method for preservation of bacteriophage MS2.3. We isolate bateriophage from Sewage, industrial sewage and sea water. Their bacteriophage plaques are different in size and shape. The survival rates of the bacteriophage MS2 which have been added to the different water bodies are different in different water environments and under different temperatures. The surviving time of bacteriophage MS2 under 4℃is longer than that under the room temperature. Bacteriophage MS2 show higher resistance to tap water and ultrapure than the other water environments.4. O-phthalaldehyde (OPA) show high performance on inactivation to bacteriophage MS2 and time is the main factor in its inactivation action. To bacteriophage MS2, in 1500mg/L of OPA solution, for a contact time of 3min, or 2000mg/L for 2min, or 2500mg/L for 1min, it could reach the disinfection level (LIV≥4.00 log₁₀).SECOND PART: Preliminarily study on the inactivation mechanism of O-phthalaldehyde (OPA) to bacteriophage MS2.1. To bacteriophage MS2, in 500mg/L of OPA solution, for a contact time of 3min, or 5000mg/L for 0.5min, particles are breakdown and adsorbtion property disappear on the whole and illustrate maturation protein is disrupted; To bacteriophage MS2, in 500mg/L of OPA solution, for a contact time of 3min, or 1000mg/L for 0.5min, or 5000mg/L for 0.5min, antigenicity disappear and illustrate coat protein is disrupted;2. To bacteriophage MS2, in 500 mg/L of OPA solution, for a contact time of 3min, or 5000mg/L for 0.5min, four genes of bacteriophage MS2 are not amplified and illustrate genes of coat protein,lysis protein, assembly protein A and RNA-dependent RNA polymerase are all disrupted. ConclusionThe genetic stability of bacteriophageMS2 retain well. Stocking under 4℃condition is a good method for preservation of bacteriophage MS2. Bacteriophage MS2 show higher resistance to tap water and ultrapure water than the other water environments. O-phthalaldehyde (OPA) show high performance on inactivation to bacteriophage MS2. The four genes of MS2, coat protein,lysis protein, assembly protein A and RNA-dependent RNA polymerase are all destroyed, particles are breakdown, adsorbtion to F-pilus of host disappear on the whole and antigenicity disappeared after it being inactivated by OPA.