摘要
目的 对牡蛎中诺如病毒检测的三种前处理方法进行比较并探讨应用策略.方法 2014年8月-2015年3月采集160只市售牡蛎,每组5只,共32组样品,取其肠腺组织采用直接处理法（M1）、PEG 8000沉淀法（M2）和蛋白酶K消化-PEG 8000沉淀法（M3）三种方法进行前处理,提取核酸后荧光定量RT-PCR检测诺如病毒核酸.应用串并联法对三种前处理方法的样品阳性率进行分析,利用x2检验比较不同方法的阳性率差异,应用一致性检验比较不同方法的一致性,对荧光RT-PCR所得到的Ct值进行ANOVA检验.结果 M1、M2、M3三种前处理方法诺如病毒阳性率依次为15.63％（5/32）、34.38％ （11/32）和37.50％ （12/32）;M1/M2/M3并联使用的阳性率最高,为50.00％ （16/32）;其次为M2/M3并联法,阳性率为46.88％ （15/32）;M1＋ M3串联或M1＋ M2＋ M3串联的阳性率最低,均为9.38％ （3/32）.M2/M3与M1/M2/M3的阳性率经x2检验比较,差异无统计学意义（P＞0.05）,两种方法经一致性检验,kappa=0.931（P＜0.05）,一致性较高.M1、M2、M3三种方法检测阳性样品的Ct值依次为33.44±0.66、33.70±1.88和33.42±2.44.经ANOVA检验,差异无统计学意义（P＞0.05）.结论 常规监测时推荐使用M2或M3,暴发疫情溯源时可考虑使用M2/M3并联法.
Objective To compare three pretreatment methods of oyster for the detection of norovirus and to discuss the application strategy.Methods From August 2014 to March 2015,160 oysters were collected and were divided into 32 groups of five.Intestinal glands were pretreated with direct treatment （M1）,PEG （polyethylene glycol） 8000 precipitation （M2） and proteinase K digestion-PEG 8000 precipitation （M3）.Nucleic acid of norovirus was extracted and detected by real time RT-PCR.The positive rates of three pretreatments were analyzed using parallel test or serial test.The positive rates between different groups were compared by x2 test and consistency test.ANOVA was applied to compare the Ct values in real time RT-PCR.Results The positive rates of norovirus of M1,M2 and M3 were 15.63% （5/32）,34.38% （11/32） and 37.50% （12/32）.Parallel test of M1/M2/M3 showed the highest positive rate of 50.00% （16/32）,followed by parallel test of M2/M3 with a positive rate of 46.88% （15/32）.The positive rates in serial tests of M1 ＋ M3 or M1 ＋ M2 ＋ M3 was the minimum of 9.38% （3/32）.The positive rates in parallel tests of M2/M3 and M1/M2/M3 showed no significant difference by x2 test （P 〉 0.05）,and these two methods showed the high consistency with kappa =0.931 （P 〈0.05）.The Ct values of M1,M2 and M3 by real time RT-PCR were 33.44 ±0.66,33.70 ± 1.88 and 33.42 ±2.44,and no statistical significance was found using ANOVA （P 〉 0.05）.Conclusions The Pretreatment methods M2 or M3 are suitable for routine surveillance,and the parallel test of M2/M3 is recommended for the tracing of norovirus outbreak.
出处
《国际病毒学杂志》
2015年第3期153-157,共5页
International Journal of Virology
基金
北京市自然科学基金项目（7132045）,食物中毒诊断溯源技术北京市重点实验室开放基金（2014BZ0063-01）
