Preliminary Study On Inactivation Mechanism And Effectiveness Of Sodium Dichloroisocyanusate To Bacteriophage MS2

Objective:To provide the furthermore theoretical and experimental for the bacteriophage MS2 in substitution of poliovirus type 1 as indicator virus in evaluation of disinfection efficacy. We observed the fundamental characteristics of bacteriophage MS2, the inactivation effectiveness and mechanism of sodium dichloroisocyanurate (NaDCC) to this bacteriophage in laboratory.Methods:There are two parts including in this research:1. The research of biological characteristices for the bacteriophage MS2:1.1 For one-step growth experiments,following centrifugation at 10000r/min for 1 min after 15 min adsorption,the pellet containing (partially) infected cells was resuspended in 5 ml ofpre-warmed nutrient broth and incubated with 160r/min at 37℃.Samples (300μl) were taken at every time (up to 3h)and immediately tittered by t, he double-layer agar plate method. Experiments were carried out in triplicate.1.2 In the experiment of optimal muhiplicity of infection (MOI), According to the method of Lu etc.the host bacterias were infected with MS2 at eight different MOI (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1000), then incubated for 4h at 37℃, and the phage titer of supematant was determined. Select the optimal MOI.1.3 In the study about host bacterials, assessing the cleavage of the phage MS2 for each test organism through the double-agar-layer plaque technique. Meanwhile, we observed the status and quantity of the plaque and selected the hosts of the MS2 phage among test organisms.1.4 The virucidal activity of sodium dichloroisocyanusate against bacteriophage MS2 is assessed by suspension test. The neutralizer is selected and appraised by test of neutralizer. Bacteriophage MS2 Was detected and enumerated by the double-agar-layer plaque technique. The experiment is designed according to Technical Standard for Disinfection (2002).2 The research of inactivation mechanism of NaDDC to bacteriophage MS2:2.1 The changes of gene of phage MS2 after inactivated by NaDDC: Using software prime premier to design primer and choose the suitable primes to RT-PCR and detect the four genes of phage MS2.2.2 The changes of proteins of phage MS2 after inactivated by NaDDC: By using transmission electron microscope, learn the change of morphous and adsorbability to F-pilus of MS2. Serum neutralization test (SNT) is operated to observe the antigen of MS2.Results:1 The research of biological characteristices for the bacteriophage MS2:1.1 One-step growth curve for MS2 shows that the latent period appears at 20 rain, the rise period at 90min, and the average burst size is about 77 pfu/cell.1.2 The optimal MOI of MS2 is between 0.01and 0.001.1.3 Among the test organisms, the hosts of the MS2 phage are E-coil ATCC 15597, E-coil 285 and E-coil B. And the susceptive order of MS2 phage for the former three hosts from the greatest to the smallest is probably as following: ATCC 15597, E-coil 285 and E-coil B.1.4 A Study on the Resistance of BacteriophageMS2 toNaDDC:With 500mg/L ofNaDDC,within a contact time of 20min; or 600mg/L, 5min, or 700rag/L, 5min, the LIV of bacteriophage MS2 can achieve the "disinfection" level;2. The research of inactivation mechanism of NaDDC to bacteriophage MS2:2.1 The changes of gene of phage MS2 after inactivated by NaDDC:With 600 mg/L of NaDDC, within a contact time of 10rain, or 800mg/L, 5min, none sequence is detected in each of the four genes from PCR;2.2 The changes of proteins of phage MS2 after inactivated by NaDDC: by the electron microscope, fragments of phage MS2 were observed, and MS2 is no adsorbability to F-pilus. The result of SNT is that the antigen of MS2 is broken.Conclusions:1 The research of biological characteristices for the bacteriophage MS2:1.1 The latent time which bacteriophageMS2 infects host Escherichia coli ATCC 15597 is short.1.2 The optimal muhiplicity of infection (MOI) of bacteriophageMS2 is between 0.01 and 0.001.1.3 The affectability ofphageMS2 to its host Escherichia coli ATCC 15597 is highest. The bacteriophageMS2 can be inactivated by sodium dichloroisocyanusate.1.4 NaDDC is efficient to bacteriophage MS2.2. The research of inactivation mechanism of NaDDC to bacteriophage MS2:2.1 The genes ofbacteriophageMS2 all were destroyed;2.2 The changed shape and fragments of its capsid were observed, its adsorption to F-pilus of host and antigenicity disappeared after it being inactivated by NaDDC.