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本研究旨在建立一种快速、准确的禽流感病毒灭活效果评价方法。传统方法通过鸡胚繁殖或细胞培养对禽流感病毒灭活效果进行定量检测，但存在操作时间长且无法客观反映残余病毒量的局限性。实时荧光定量逆转录PCR(RT-qPCR)虽可作为直接定量消毒剂作用后的残余病毒量的一种方法，但无法评估病毒的感染性。本研究将RT-qPCR技术与酶法预处理相结合，对中低效消毒剂新洁尔灭和75%乙醇灭活H9N2病毒的效果进行评估。结果显示，333 U/mL的蛋白酶K处理30 min，可有效降解受损的病毒囊膜蛋白；随后使用0.2 mg/mL的RNase A处理30 min，可降解裸露的病毒RNA。1%和0.25%的苯扎溴铵分别作用5 min后，经酶法预处理联合RT-qPCR检测，病毒基因组水平分别降低5.67和5.62 lg拷贝数/μL;75%乙醇作用1 min，病毒基因组水平降低5.65 lg拷贝数/μL；作用时间延长至5 min时，病毒基因组水平降低至检测值以下。本研究通过RT-qPCR联合酶法预处理建立了定量评价苯扎溴铵和75%乙醇有效灭活H9N2禽流感病毒的方法 。
Abstract：This study aimed to develop a rapid and accurate method for evaluating the inactivation efficacy ofavian influenza virus(AIV). Traditional approaches, such as viral propagation in embryonated chicken eggs orcell cultures, are time-consuming and fail to objectively quantify residual viral particles. Although real-timequantitative reverse transcription PCR(RT-qPCR) allows direct quantification of residual viral RNA followingdisinfection, it does not distinguish between infectious and non-infectious virus. To overcome this limitation, wecombined RT-qPCR with enzymatic pretreatment to assess the inactivation efficiency of two commonly useddisinfectants—benzalkonium bromide and 75% ethanol—against H9N2 AIV. Treatment with 333 U/mL ofproteinase K for 30 minutes effectively degraded compromised viral envelope proteins. This was followed bytreatment with 0.2 mg/mL of RNase A for 30 minutes, which degraded exposed viral RNA. After 5 minutes ofexposure, 1% and 0.25% benzalkonium bromide reduced viral genome copy numbers by 5.67 and 5.62 log10copies/μL, respectively, as determined by RT-qPCR following enzymatic pretreatment. Exposure to 75%ethanol for 1 minute reduced the viral genome level by 5.65 log10 copies/μL, and extended treatment for 5minutes reduced viral RNA to below the detection limit. These findings demonstrate that combining enzymaticpretreatment with RT-qPCR provides a quantitative and effective method for evaluating the inactivation ofH9N2 AIV by benzalkonium bromide and 75% ethanol.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250063
中图分类号:S852.65
引用信息:
[1]王慧,王明明,张杰琼等.实时荧光定量PCR与酶法预处理联合应用对H9N2禽流感病毒灭活效果评价的初步研究[J].病毒学报,2025,41(03):676-682.DOI:10.13242/j.cnki.bingduxuebao.250063.
基金信息:
“十三五”国家科技重大专项（项目号：2018ZX10201002）,题目：基于海量多元大数据的突发急性传染病时空多尺度预测预警模型与应用示范的构建~~