贝类中甲型肝炎病毒磁珠免疫沉淀提取方法的建立及其与国标方法的比对

Establishment of dynabeads immunoprecipitation extraction method of hepatitis A virus from shellfish and its comparison with national standard method

目的建立蛋白酶K结合磁珠免疫沉淀提取贝类中甲型肝炎病毒(hepatitis A virus,HAV)的方法,并与国际标准方法(ISO15216—2017)进行比较研究。方法应用高效价的HAV单克隆抗体,采用偶联技术,通过优化参数,建立蛋白酶K结合磁珠免疫沉淀的提取方法;通过人工预加入103~105 CCID50/mL的4种甲型肝炎活病毒,计算回收率和相对标准偏差(relative standard deviation,RSD),评价方法的提取效果;通过对来自5个产地的4种贝类进行病毒提取和一步法实时荧光定量逆转录PCR(quantitative real-time reverse transcriptase polymerase chainreaction,qRT-PCR)检测,评价方法的适用性。结果HAV单抗效价为1×107,纯度为94.36%,与戊型肝炎病毒、乙型肝炎病毒以及同为小RNA病毒的柯萨奇病毒A16型和肠道病毒A71型均无交叉反应,特异性良好。贝类样品中HAV的平均回收率为22.02%,RSD为0.67%~5.87%,表明该方法对HAV是有效的。与国际标准提取方法相比,在不同浓度下可将HAV检测拷贝数提高10倍以上,显示出良好的剂量效应关系,并且可将不同产地间、不同品种贝类间的检测RSD从4.31%~44.75%和0.61%~29.10%,分别降低至1.76%~6.90%和0.63%~4.80%,有效降低贝类组织中干扰成分的影响。结论该方法较国际标准方法回收率高、抗干扰能力强,具有良好的重现性,提高了贝类等食品中HAV检测灵敏度、可靠性,为贝类等食品安全检测提供了关键的提取手段。

Objective To establish a method for extracting hepatitis A virus(HAV)in shellfish by proteinase K combined with dynabeads immunoprecipitation and comparing it with the international standard method(ISO 15216—2017).Methods Amonoclonal antibody(mAb)of HAV with high titer was used to establish an optimized method relying on proteinase K combined with dynabeads immunoprecipitation and coupling technology.Four strains of HAV with dosage of 103-105 CCID50/mL were added to shellfish manually,and the recoveries and relative standard deviations(RSDs)were calculated by using established method to evaluate extractive ability.HAV in 4 kinds of shellfish from 5 producing regions was extracted and detected with quantitative real-time reverse transcriptase polymerase chain reaction(qRT-PCR),to evaluate applicability of evaluation method.Results Titer of HAV mAb was 1×107,and the purity was 94.36%.Cross-reaction with hepatitis E virus,hepatitis B virus,coxsackie virus A16 and enterovirus A71 which were also picornavirus was detected,meaning the specificity was good.Average recoveries of HAV in samples were 22.02%,RSDs were 0.67%-5.87%,indicating the method was effective for those HAV viruses.Copies of HAV was increased with more than 10 times compared with ISO 15216—2017 by using various concentrations of HAV,which showed good dose-dependent relationship.The coefficients of variation of shellfish from different habitats and varieties reduced from 4.31%-44.75%and 0.61%-29.10%to 1.76%-6.90%and 0.63%-4.80%respectively,which effectively reduced the influence of interference components in shellfish tissues.Conclusion Compared with the international standard method,the method has high recoveries,strong anti-interference ability and good reproducibility.It improves the sensitivity and reliability of HAV detection for shellfish and other foods,and provides a key method for food safety detection of shellfish and other foods.