反转录PCR技术检测贝类中诺瓦克样病毒的研究

目的:从病毒富集和核酸提取两方面进行探索,旨在建立一个反转录(RT)PCR技术检测贝类中诺瓦克样病毒(NLVs)的方法.方法:利用脊髓灰质炎病毒作为参照毒株,优化甘氨酸缓冲液一聚乙二醇(PEG)病毒浓缩方法;同时比较异硫氰酸胍法、SDS一蛋白酶K法、Trizol-异丙醇法、试剂盒法4种RNA提取方法:对市售贝类样品进行了检测,并利用基因测序对阳性样品进行验证.结果:采用pH 9.5甘氨酸缓冲液-16%聚乙二醇病毒浓缩法,病毒的回收率为16.8%;利用Trizol-异丙醇法提取RNA,检出限为8.1×102 RT-PCR50/5gg贝肉;实际检测贝类样品25件,其中3件样品为阳性,基因测序结果亦证实为阳性.结论:建立了一个灵敏度较高的RT-PCR检测贝类中诺瓦克样病毒的方法.     

RT-PCR方法检测贝类中诺沃克样病毒的研究

目的:本研究从病毒富集和核酸提取两方面进行探索,旨在建立一个反转录(RT)PCR技术检测贝类中诺沃克样病毒(NLVs)的方法.方法:利用脊髓灰质炎病毒作为参照毒株,优化了甘氨酸缓冲液-聚乙二醇(PEG)病毒浓缩方法;同时比较了异硫氰酸胍法、SDS-蛋白酶K法、Trizol-异丙醇法、试剂盒法四种RNA提取方法;对市售贝类样品进行了检测,并利用基因测序对阳性样品进行验证.结果:本研究采用的pH9.5甘氨酸缓冲液 -16%聚乙二醇病毒浓缩法,病毒的回收率为16.8%,利用Trizol-异丙醇法提取RNA,检出限为8.1×10 2 RT-PCR50/5g贝肉,实际检测贝类样品25件,其中3件样品为阳性,基因测序结果亦证实为阳性.结论:本研究建立了一个灵敏度较高、较为有效的RT-PCR技术检测贝类中诺沃克样病毒的方法.     

数字聚合酶链式反应法检测贝类和浆果中甲肝病毒

目的建立一种数字聚合酶链式反应(PCR)检测贝类和浆果中甲肝病毒的方法。方法样品经蛋白酶K消化-聚乙二醇法进行甲肝病毒富集后,使用高纯度病毒核酸试剂盒进行RNA提取,之后对甲肝病毒进行数字PCR检测。结果本方法对甲肝病毒有典型扩增,重复性和稳定性良好,对于草莓样品中甲肝病毒的检测灵敏度为25.30 CCID_(50)/20 g,树莓样品中甲肝病毒的检测灵敏度为6.32 CCID_(50)/20 g,贝类样品中甲肝病毒的检测灵敏度为12.54 CCID_(50)/2 g,表明其灵敏度高。结论该方法快速、准确、灵敏,适合测定贝类和浆果食品中甲肝病毒。     

RT-PCR法检测贝类中的甲肝病毒的研究

在世界范围内,甲肝病毒是与食用贝类有关的主要传染性疾病之一。由于贝类中含有PCR抑制剂以及病毒富集过程中病毒的回收率低,阻碍了天然污染的贝类中HAV的PCR检测。研究中建立了一种经苷氨酸缓冲液洗涤,2次PEG沉降富集病毒,然后进行RNA提取和RT-PCR对贝类中的甲肝病毒进行检测的方法。经比较,采用小体系肠道样品检测比采用全贝检测的富集效果更佳,并比较了PEG8000和PEG6000对病毒富集的效果,回收率分别为13.5%和7.6%,此方法可有效地降低PCR抑制剂的影响,最低检测限可达10个TCID50/1.5 g。     

草莓中甲型肝炎病毒检测

目的：建立草莓中甲型肝炎病毒（hapatitis A virus,HAV）的有效富集方法及病毒RNA提取方法,用于草莓中HAV检测。方法：利用甲肝减毒疫苗对已知阴性的草莓样品进行人工污染,通过有效富集条件的优化及RNA提取后,采用实时荧光逆转录-聚合酶链式反应进行检测。结果：病毒富集选择Tris-甘氨酸-1 g/100 mL牛肉浸提物缓冲液洗脱、果胶酶30 U、氯仿-正丁醇为抑制剂去除剂、聚乙二醇沉淀、5 ℃孵育1 h等优化条件,检测灵敏度较高;最优RNA提取方法为美国ABI公司生产的RNA提取试剂盒。采用优化后的方法对草莓样品中HAV病毒的检测显示,该病毒粒子的检测灵敏度可以达31.36 CCID50/20 g样品。同时对50 份送检样品进行检测,结果均为阴性。结论：所建立的病毒富集方法和核酸提取方法更适合草莓样品中HAV的检测,灵敏度较高。     

蔬菜及水果中NLVs和HAV检测的研究

目的建立蔬菜水果中诺沃克样病毒(NLVs)和甲肝病毒(HAV)的RT-PCR检测方法。方法使用具有较强缓冲力的甘氨酸缓冲液将病毒从试样表面洗脱下来,用PEG6000将病毒沉降后进行RNA提取和RT-PCR检测。结果蔬菜中NLVs和HAV检测的灵敏度可达1.0×103RT-PCRU50/30 g试样和30 TCID50/30 g试样。而草莓样品的NLVs和HAV最低检出限为1.7×103RT-PCRU50/100 g试样和48 TCID50/100 g试样。结论该方法灵敏、可靠。     

实时荧光RT-PCR检测贝类中的札幌病毒

建立检测贝类中GⅠ、GⅡ、GⅣ和GⅤ型札幌病毒的实时荧光RT-PCR新方法。首先使用PEG 8000对贝类中的札幌病毒进行富集,然后采用Tri-reagent提取材料中的总RNA,针对札幌病毒RNA 3'端含Poly A尾的特点,使用带有Poly(dT)25的磁珠对病毒RNA进行纯化,用所获的高纯度RNA进行四种型别札幌病毒的实时荧光RT-PCR检测。该方法高效、灵敏,检测下限为101数量级拷贝,能够用于日常检验。     

食品中甲型肝炎病毒RNA提取方法的比较及应用

目的：对食品中甲型肝炎病毒的3 种RNA提取方法进行综合比较,以优选出最佳的核酸提取方法,为各级实验室开展食源性甲型肝炎病毒核酸检验提供技术参考。方法：分别采用ABI AM1836、QIAGEN 74104和 ROCHE11858882001三种商业化核酸提取试剂盒对人工污染甲型肝炎病毒的食品样品进行核酸提取,根据3 种核酸提取方法的提取效率、抑制剂去除效率、检测灵敏度、综合应用指标进行病毒RNA的优化,且采用优化后的提取方法进行实际样品的检测。结果：综合病毒各项评价指标显示,ROCHE 11858882001在食品中提取甲型肝炎病毒RNA效果最优,其次为QIAGEN 74104和ABI AM1836。101 份样品的检测结果显示,一份蓝靛果样品为甲型肝炎病毒核酸阳性,其余样品均为阴性。结论：ROCHE 11858882001是一种快捷有效的病毒RNA提取方法,适用于食品中甲型肝炎病毒的日常检测工作。     

北京市市售贝类、蔬菜、浆果、即食海产品中诺如病毒污染状况检测及检测方法探析

目的对北京市市售贝类(牡蛎、贻贝、扇贝、文蛤、毛蚶)、蔬菜(苗菜、生菜)、浆果(草莓、蓝莓)、即食海产品(三文鱼、即食虾)进行诺如病毒检测。方法贝类、蔬菜、浆果类样品参照ISO/TS 15216-1方法进行前处理,虾类检测参照贝类、三文鱼参照蔬菜的方法并进行改良。病毒RNA提取参照罗氏High pure viral RNA Kit试剂盒方法对样品前处理后的提取液进行提取。实时荧光逆转录聚合酶链反应(实时荧光RT-PCR)的检测使用罗氏Light Mixnoroviurus GI-GII于Lightcycler480 II进行检测,并进行GI-GII分型。诺如病毒阳性样品,参照SN/T2626—2010《国境口岸诺如病毒检测方法》,使用JV12、JV13进行扩增,将获得的DNA扩增片段进行测序并分型。结果经罗氏试剂盒检测贝类食品478份(40份阳性)、蔬菜62份(1份阳性)、浆果84份(0份阳性)和即食海产品51份(1份阳性)。有42份为GI-GII,其中37份为GII型,4份为GI型。在42份阳性样品中,有29份GII型阳性样品对RNA聚合酶区域扩增成功,通过序列分析,27份为GII.17,1份为GII.12,1份为Hawaii.calicivirus。结论通过对北京市市售食品样品中诺如病毒检测发现,贝类食品呈现较明显的季节分布,在冬季检出率较高,贝类食品是诺如病毒污染率最高的食品。     

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.     

Detection of noroviruses in foods: a study on virus extraction procedures in foods implicated in outbreaks of human gastroenteritis

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation-RNeasy-Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.     

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.