| Since the end of 2019,Corona Virus Disease 2019(COVID-19)has swept the world.The quantity of infectious waste has increased dramatically,posing a serious threat to people’s health and pandemic prevention and control.Disposing of infectious waste in time,safely and professionally is an important link to prevent secondary transmission and block the spread of the pandemic.COVID-19 pandemic has brought many challenges to the disposal of infectious waste,such as sharp increase in quantity,damage and leakage during transportation,risk area and personnel control.Therefore,it is necessary to strengthen the research on rapid in-situ disinfection technology of infectious waste.As a physical disinfection method,microwave disinfection is a combination of thermal effect and non-thermal effect.Compared with other disinfection methods,microwave disinfection has the characteristics of fast,strong penetration ability,consistent temperature rise,energy conservation,high efficiency,convenience,economy and environmental protection.It is especially suitable for the in-situ harmless treatment of infectious waste.At present,the quantitative evaluation of microwave disinfection effect on virus is very few,mainly focusing on bacteria,and the inactivation mechanism is not clear.Viral respiratory infectious diseases have the characteristics of frequent gene mutation,easy transmission,strong infectivity,and general susceptibility.They are very easy to cause outbreaks and epidemics.Therefore,with the pandemic of COVID-19,it is particularly important to research the inactivation effect of microwave on respiratory virus in infectious waste and establish microwave disinfection technology for rapid in-situ harmless treatment.Considering the limitation of biosafety level,this study selected measles virus(MV)as the representative of respiratory RNA virus,simulated infectious waste with masks,gloves and swabs commonly used during epidemics,researched the inactivation effect of microwave and its influencing factors,and discussed the molecular mechanism of microwave disinfection,so as to provide scientific basis for the application of microwave disinfection technology to the rapid in-situ harmless treatment of respiratory virus in infectious waste.To sum up,it has important practical significance for the prevention and control of viral respiratory infectious diseases.ObjectiveTo research the inactivation characteristics,regularities and molecular mechanism of microwave disinfection on infectious waste carrying respiratory RNA virus,so as to provide scientific basis for the development of rapid in-situ harmless microwave disinfection technology on infectious waste.Methods1.Preparation of virus suspension and determination of virus titerMeasles virus was inoculated into Vero-SLAM cells,incubated at 35℃ in 5%CO2 until cytopathic extent reached 75%,subjected to three freeze-thaw cycles,centrifuged and harvested the virus supernatant.Fifty percent cell culture infectious dose(CCID50)of MV was measured by 50%endpoint method.2.Preparation of simulated infectious wasteDripping 25 μL of MV suspension onto sterile mask samples(3 cm × 3 cm),glove samples(3 cm × 3 cm)and swabs to simulate infectious waste contaminated by MV.3.Setting of microwave conditions and treatmentPlacing simulated infectious waste in a fixed position in the microwave device,the output power included 300 W,500 W and 700 W,which was treated for 30 sec,60 sec and 90 sec respectively.When the power was 300 W and mask was carrier,take it as the general group,and add humidity increasing group and wrapped group of infectious waste.In the humidity increasing group,25 μL of virus suspension and 75 μL of cell maintenance medium was mixed evenly and then dropped on the mask sample.In the wrapped group,a complete mask is wrapped on the outer layer of the mask sample with 25 μL of virus suspension.At the same time,the temperature change during microwave treatment was recorded and the virus control without microwave treatment was set up.After microwave treatment,the carrier was put into 5 mL of cell maintenance medium,mixed by vortex for 30 sec,then the supernatant was collected and centrifuged at 3000 × g for 10 min.The same operation was performed on the virus control group.4.Effect of microwave inactivation on MV(1)Killing rateThe supernatant collected after microwave treatment was diluted 10 times and inoculated into Vero-SLAM cells.The CCID50 of MV was measured by 50%endpoint method,and the killing rate was calculated as follows:killing rate(%)=[1-(experimental group CCID50/virus control group CCID50)]× 100%.(2)Syncytial reduction rateThe cytopathic effect(CPE)of Vero-SLAM cells infected by MV is that cell-cell fusion form syncytia.Inoculating the supernatant collected after microwave treatment into VeroSLAM cells and incubating for 36 hours,the number of syncytia were counted after Giemsa staining,and the syncytial reduction rate was calculated as follows:syncytial reduction rate(%)=[1-(the number of syncytia in experimental group/the number of syncytia in the virus control group)]× 100%.(3)Virus proliferationInoculating the supernatant collected after microwave treatment into Vero-SLAM cells,the virus proliferation after 48 hours culture was detected by quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)to quantitatively evaluate the inactivation effect of microwave on MV.5.Effect of microwave on MV geneVirus RNA was extracted from the supernatant collected after microwave treatment,and the N gene,P gene,M gene,F gene,H gene,and L gene of MV were amplified by reverse transcription polymerase chain reaction(RT-PCR)to detect the damage of microwave on virus gene.6.Inactivation of MV by heat treatmentThe highest temperature in the process of microwave treatment was selected to heat MV for 30 sec,60 sec and 90 sec respectively.The virus activity was detected,and genes of MV were amplified by RT-PCR.The results were compared with microwave treatment to further explore the inactivation mechanism of microwave on MV.7.Data analysisThe data were statistically analyzed by SPSS 26.0.The experiment was repeated three times.The lgCCID50 and Ct values of each group were compared by t-test and one-way analysis of variance(ANOVA).Pearson correlation analysis was carried out between killing rate and syncytial reduction rate,and the Bland-Altman diagram was drawn for consistency evaluation(α=0.05)。Results1.MV titerThe titer of MV was 105.7 CCID50/0.1mL determined by 50%endpoint method.2.Temperature change caused by microwaveMicrowaves could cause temperature rise.Increasing microwave power,treatment time or humidity of infectious waste,the temperature increment caused by microwave was also higher and the maximum temperature was 80℃ at 700 W for 90 sec.The experimental group with high microwave power or long treatment time had a temperature drop after reaching the maximum temperature,and the change of temperature varied with the type of carriers.3.Killing effect of microwave on MV and influencing factorsThe results of the killing rate showed that different carriers had different killing effects.The killing rate increased with the increase of microwave power or time,indicating that increasing power or treatment time could enhance the inactivation effect of microwave on MV.With mask as carrier,the killing rate exceeded 99.61%when the microwave condition was 500 W,90 sec or 700 W,30 sec;with glove as carrier,the killing rate exceeded 99.50%when the microwave condition was 500 W,30 sec;with swab as carrier,the killing rate exceeded 97.65%when the microwave condition was 500 W,90 sec or 700 W,30 sec.In the same irradiation time,the killing rate of humidity increasing group and wrapped group were higher than that of general group,which meant increasing humidity or sealability of infectious waste could enhance the inactivation effect.4.Effect of microwave on the syncytial reduction rate and influencing factorsThe results of syncytial counting showed that there were different syncytial reduction rates on three carriers,but they all increased with the increase of power or time.At 700 W for 90 sec,syncytial reduction rates all reached 100%on three carriers.The syncytial reduction rate could be enhanced by increasing humidity or sealability of infectious waste.5.Consistency evaluation of the killing rate(%)and the syncytial reduction rate(%)We evaluated the consistency between the killing rate(%)and the syncytial reduction rate(%)under the same microwave condition using Pearson correlation analysis and Bland-Altman analysis,which showed a strong correlation(r>0.9,P<0.001)and good consistency(H0:Mean=0,P>0.05).6.Effect of microwave on the proliferation of MV and influencing factorsThe results of qRT-PCR showed that compared with virus control,the higher microwave power or the longer time was,the higher Ct value of experimental group was,and the difference was statistically significant(P<0.05).Compared with virus control,the difference of Ct value in the general group at 60 sec was statistically significant(P<0.05),while the difference of Ct value in the humidity increasing group or the wrapped group at 30 sec was statistically significant(P<0.05).It indicated that increasing microwave power,treatment time,humidity and sealability of infectious waste could enhance the ability of microwave to inhibit MV proliferation.7.Damage of MV gene by microwave irradiationRT-PCR results showed that microwave could damage N gene,P gene,M gene,F gene,H gene,and L gene of MV,and the higher microwave power or the longer time was,the more serious the damage was.8.Inactivation effect of MV by heat treatmentThe maximum temperature of 80℃ during microwave treatment was selected as the temperature to heat MV separately.The results showed that the killing rate of MV treated at 80℃was lower than that of microwave disinfection with the power of 500 W and 700 W in the same time.Furthermore,heat treatment at 80℃ for 90 sec could not damage MV gene.Conclusions1.Microwave power of 700 W for 90 sec can inactivate more than 99.99%of 25 μL of MV with the titer of 105.7 CCID50/0.1mL on simulated infectious waste with masks,gloves and swabs as carriers.The inactivation effect of microwave on MV is affected by the type of carrier,positively correlated with microwave power and treatment time,enhanced with the increase of the humidity and sealability of infectious waste.2.The inactivation effect of microwave on MV is higher than that of heat treatment.Its mechanism is related to the non-thermal effect of microwaves to damage the MV gene,and the degree of damage is positively correlated with microwave power and treatment time. |
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