TRIzol（或TriPure）试剂结合QIAamp Viral RNA Mini试剂盒用于血标本中病毒RNA提取的研究

目的 建立一种安全、简便、可靠的适用于血标本中有包膜RNA病毒核酸提取的方法,保证埃博拉病毒核酸筛查的实验室安全,提高标本的核酸检测效率.方法 根据已发表可完全灭活埃博拉病毒的方法,通过实时荧光逆转录聚合酶链反应（real-time RT-PCR）方法,比较分析QIAamp Viral RNA Mini试剂盒、TRIzol（或TriPure）试剂裂解样本并用或不用氯仿萃取后结合QIAamp Viral RNA Mini试剂盒等3种方法提取核酸的效率,优化反应步骤,建立一种安全、简便的血标本中病毒RNA提取方法.结果 血标本经TRIzol（或TriPure）试剂处理后,不经氯仿萃取,与QIAamp Viral RNA Mini试剂盒结合,提取病毒RNA,RNA提取效率优于QIAamp Viral RNA Mini试剂盒和美国CDC推荐的方法.结论 采用TRIzol（或TriPure）试剂裂解病毒,结合QIAamp Viral RNAMini试剂盒提取病毒RNA的方法简便、可靠,可用于埃博拉出血热相关标本的核酸检测.     

EV71、CA16及通用型肠道病毒实时荧光定量PCR诊断试剂盒的研发

目的 研制一种新的含竞争性内标引物,能同时检测EV71、CA16及通用型肠道病毒的四重实时荧光定量PCR诊断试剂盒,用于手足口病的临床诊断及流行病学监测.方法 设计特异性的EV71型、CA16型及其他型肠道病毒和内参基因的引物,改进病毒核酸提取方法,优化实时荧光定量PCR检测体系,并研究产品的准确度、稳定性、精密度和扩增效率和检测线性范围.结果 本试剂盒的引物和探针特异性好,试剂盒采用的的病毒RNA抽提效果与QIAGEN公司QIAamp Viral RNAmini Kit抽提效果相当,但具有更低的试剂成本.优化引物探针浓度分别为0.2μmol/L的上下游引物浓度、0.06 μmol/L的通用探针浓度、0.08 μmol/L的EV71分型探针浓度和0.08μmol/L的CA16分型探针浓度.产品具有较好的稳定性和良好的准确度、精密度,CA16、EV71和肠道病毒通用型对引物的扩增效率分别是106％、101％和105％,线性检测范围是109拷贝/μl ～ 102拷贝/μl.结论 采用四重荧光定量PCR技术开发的能同时检测EV71、CA16、其他肠道病毒和内标的手足口病诊断试剂盒具有良好的准确性、稳定性、精密度和扩增效率等,具有很好的临床应用价值.     

不同方法提取SD大鼠膝关节软骨组织的总RNA

背景:目前文献报道的软骨RNA提取有多钟方法,国内文献报道多为经典Trizol法,在实验中发现该方法提取的RNA并不能满足后续实验的需要。目的:探索不同方法提取SD大鼠软骨组织总RNA的区别。方法:选取3月龄SD大鼠9只,分别采用Rneasy Lipid Tissue Kit、RNAout试剂盒和经典Trizol法对软骨组织进行总RNA提取,并通过RNA琼脂糖凝胶电泳检测RNA的完整性,以探寻软骨总RNA最佳提取方案。结果与结论:Trizol法提取的RNA A260/A280值为1.58,说明纯度不高;RNAout法提取的RNA,由于软骨含有大量的蛋白多糖和胶原,也不能满足后续实验的需要;RNeasyMini Kit法和肝(Trizol)法提取RNA的A260/A280值分别为2.00和1.98,说明提取的总RNA或核酸的纯度高。RNA的完整性电泳结果显示,RNeasyMini Kit法、RNAout法和肝(Trizol)法可见28 s、18 s及5 s条带,而Trizol法未见任何条带,RNAout法28 s和18 s条带不清楚。结果提示Rneasy Lipid Tissue Kit法获得的总RNA纯度高、完整性和稳定性好,并能成功反转录合成双链c DNA,而RNAout试剂盒和经典Trizol法获得的总RNA不能满足后续实验需要。     

四种腺病毒核酸提取方法的比较

目的通过蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种腺病毒核酸提取方法的比较。为进一步研究腺病毒的分子生物学特性选择合适的方法提供参考。方法以腺病毒感染的A549细胞为样品．分别以蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸．核酸经紫外分光光度计检测A260／A280的比值后,用荧光定量PCR方法检测核酸中腺病毒拷贝数浓度。并记录四种腺病毒核酸提取方法所需的操作时间。结果蛋白酶K法、苯酚法、病毒DNA／RNA提取试剂盒、腺病毒荧光PCR检测试剂盒等四种方法提取核酸的A260／A280的比值依次为1．85532、1．7377、1．81474和1．43934,核酸中腺病毒拷贝数浓度依次为4．9×10^5copies／mL、3．94×10^3copies／mL、2．66×10^6copies／mL和6．15×10^6copies／mL,提取核酸所需的时间分别为1．5、15、0．5和0．5h。结论四种腺病毒核酸提取方法中,蛋白酶K法简单快捷、成本低廉,适合一般实验室使用;苯酚法适合用于提取细胞培养上清中病毒的核酸;病毒DNA／RNA试剂盒的优点是操作时间短,得到的病毒核酸纯度较高;腺病毒荧光PCR检测试剂盒对病毒的核酸损耗少．操作步骤少．适用于临床标本的腺病毒核酸检测。     

套式RT-PCR方法检测SARS冠状病毒

采用世界卫生组织推荐的套式RT PCR引物 ,建立了较稳定、重复性好的实验室检测方法。病理检测的肺组织 3份 ,取自广州非典死亡病例 ;咽拭子和漱口水 2 0份 ,取自广州非典临床确诊病例 ;SARS冠状病毒 ,本室保存。Vero E6、大肠杆菌DH5α为本实验室保存。提取质粒试剂盒用QIAGEN公司的QIAampViralRNAMiniKit,逆转录及第一次PCR用QIAGEN公司的QIAGENOneStepRT PCRKit。第二次PCR所用Taq酶和核酸凝胶回收试剂盒为TaKaRaTaqTM和PCRFragmentRecoveryKit,均购自宝生物工程有限公司 ,dNTP购自Pharmacia公司 ,克隆载体p…     

人精子核完全裂解对RNA提取的影响

目的 证实提取人精子RNA时,精子核完全裂解的重要性.方法 人精子液化后经Pereoll纯化,淋巴细胞分离液分离外周血自细胞用作对照.人精子和外周血白细胞RNA均用TRIzol和RNeasy Kit两种试剂提取,对于不同试剂提取的RNA,取相同细胞数的RNA用于逆转录,并用实时PCR检测两种提取方法的β-ACTIN mRNA 量,来反映所提取RNA的量.结果 RNeasy Kit能完全裂解人精子和外周血白细胞的细胞核,TRIzol能完全裂解外周血白细胞的细胞核,但不能完全裂解人精子核.对于人精子,RNeasy Kit和TRIzol提取RNA量分别为(149.8±24.5)和(35.3±4.0)ng/106精子(n=3),差异有统计学意义(P=0.01).而且这种差异不是由于提取试剂的提取效率造成的,因为对于外周血白细胞,RNeasy Kit提取RNA量还稍低于TRIzol[(765.5±229.8)和(958.8±201.0)ng/106细胞,n=3,P=0.168].结论 人精子核的完全裂解可显著增加精子RNA提取量,也间接说明精子核是人精子RNA存在的重要部位.     

人血管抑素K1-5重组腺病毒载体的构建

血管抑素K1 5是目前被认为最强的抑制血管生成的因子之一 ,研究表明其能明显抑制血管内皮细胞增生及牵移。我们采用腺病毒作为载体 ,成功构建了携带血管抑素K1 5基因的腺病毒 ,为下一步进行体内外实验奠定了基础。材料和方法 :试剂质粒pUC19及pCA13均购自加拿大MicrobixBiosystems公司 ,Trizol试剂盒及逆转录试剂盒购自美国Invitrogen公司 ,内切酶及LipofectAmine 2 0 0 0试剂盒购自美国GibcoBRL公司 ,PCR产物回收试剂盒、质粒DNA制备试剂盒和病毒DNA提取试剂盒均购自德国Qiagen公司 ,2 93细胞购自MicrobixBiosystems公司 ,正常…     

诺如病毒快速检测试剂的评价

目的评价德国R—Biopharm公司生产的RIDA QUICK诺如病毒快速检测试剂卡（免疫层析法）的准确性、安全性以及与对照试剂的等效性。方法以上市的IDEA Norovirus检测试剂盒（酶联免疫法）平行测定结果为参考,评价RIDA QUICK诺如病毒快速检测试剂卡（免疫层析法）的灵敏度、特异性及符合率。结果临床检测样本1042份,新型快速检测试剂诺如病毒检测特异性98．4％,灵敏度92．4％,与对照试剂的符合率为97．6％。结论RIDA QUICK诺如病毒快速检测试剂卡对于诺如病毒抗原的检测具有良好的特异性和灵敏度。     

两种巨细胞病毒IgG抗体ELISA诊断试剂性能评价

目的对国内两种巨细胞病毒IgG抗体ELISA诊断试剂进行评价。方法用两种试剂对BBI公司的质控盘QTC711,血清转化盘PTC901,BIOMEX公司血清转化盘SCP—CMV-001（RP-003）、SCP—CMV-002（RP-019）,儿童、孕妇和门诊患者2163份标本进行检测,对不一致的样本用DiasorinELISA试剂盒和Mikrogen重组免疫印迹试剂盒进行复测,比较两种试剂的灵敏度、特异性和对不同人群的检测差异。结果A试剂对3个血清转化盘的检出时间均早于B试剂,平均提前25d,与AbbottImxCMVIgG检测灵敏度一致。两种试剂检测孕妇样本607份,8份不一致,总符合率98．68％;门诊样本512份,7份不一致,总符合率98．63％;儿童样本1044份,74份不一致,总符合率92．91％,两试剂对儿童人群的符合率低于孕妇和门诊人群。两试剂对三个人群检测均为阴性的161份样本,和A试剂检测为阳性B试剂检测为阴性的89份样本,用意大利Diasorin公司CMV—IgGELISA试剂复测,A试剂与Diasorin的阳性符合率为100％,阴性符合率为93．06％,总符合率为95．22％;B试剂与Diasorin的阴性符合率为100％,78份阳性未检出,总符合率为68．92％。选择A试剂与Diasorin结果不一致的样本12份,一致的样本6份,用重组免疫印迹复测,其中14份阳性,4份阴性。结论国内A试剂对血清转化盘的检测窗口期较B试剂显著提前。两试剂对孕妇和门诊人群的符合率高于儿童人群。两试剂不符合的样本用进口试剂盒复测,A试剂与进口试剂盒的符合率高于B试剂,显示了较高的灵敏度。     

现场生物检材Identifiler Plus直接扩增方法的应用研究

目的探讨非直接扩增试剂Identifiler Plus Kit直接扩增现场微量生物检材的可行性。方法选取270份现场血样、68份烟蒂和194份脱落细胞擦拭物,采用IdentifilerPlus和Identifiler—Direct Kit直接扩增法及Chelex-100提取法对样本进行检测。结果PCR遗传分析数据显示IdentifilerPlus Kit直接扩增法对现场血样检测成功率为96．1％,对烟蒂及脱落细胞擦拭物检测成功率为分别为86．8％和64．9％。结论IdentifilerPlusKit能有效应用于血痕、烟蒂、脱落细胞擦拭物等现场生物检材的直接扩增。     

Efficient extraction of viral DNA and viral RNA by the Chemagic viral DNA/RNA kit allows sensitive detection of cytomegalovirus, hepatitis B virus, and hepatitis G virus by PCR

The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available.     

Direct detection of highly pathogenic avian influenza A/H5N1 virus from mud specimens

Contaminated mud and soil may play roles as reservoirs and sources of transmission for avian influenza A virus. However, the persistence of highly pathogenic avian influenza (HPAI) H5N1 virus in soil or mud has not been well documented, and specific methods of H5N1 virus detection in mud and soil specimens have not been described. The aim of this work was to evaluate the capacities of five different commercial kits and one elution-concentration technique to extract nucleic acids from H5N1 virus and to detect infectious viral particles in experimentally infected mud specimens. The viral RNA detection thresholds for the QIAamp kit, Trizol LS and the MagNA Pure LC kit were 5 × 10(2)RNA copies per gram of mud. Trizol reagent and the RNA PowerSoil? kit were unsuccessful in recovering any viral RNA from mud. When the elution-concentration technique was performed prior to nucleic acid extraction, the performance of the MagNA Pure kit increased to a level that allowed the detection of H5N1 nucleic acids in naturally contaminated environmental samples that had previously tested negative after direct extraction using commercial kits. The levels of detection of infectious virus after inoculation into embryonated eggs were higher in concentrates than in eluates.     

Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA

Automated nucleic acid extraction is an attractive alternative to labor-intensive manual methods. We compared two automated methods, the BioRobot M48 instrument (Qiagen, Inc.) and MagNA Pure (Roche Applied Sciences) methods, to two manual methods, the QIAamp Viral RNA Mini kit (Qiagen) and TRIzol (Invitrogen), for the extraction of enterovirus RNA. Analytical sensitivity was assessed by dilution analysis of poliovirus type 2 Sabin in cerebrospinal fluid. The sensitivity of PCR was equivalent after RNA extraction with QIAamp, BioRobot M48, and MagNA Pure. All 18 replicates of 100 PFU/ml were detected after extraction by the four methods. Fewer replicates of each successive dilution were detected after extraction by each method. At 10(-1) PFU/ml, 17 of 18 replicates were positive by QIAamp, 15 of 18 replicates were positive by BioRobot M48, and 12 of 18 replicates were positive by MagNA Pure; at 10(-2) PFU/ml, 4 of 17 replicates were positive by QIAamp, 2 of 18 replicates were positive by BioRobot M48, and 0 of 18 replicates were positive by MagNA Pure. At 10(-3) PFU/ml, no replicates were detected. Evaluation of TRIzol was discontinued after nine replicates due to a trend of lower sensitivity (at 10(-3) PFU/ml eight of nine replicates were positive at 100 PFU/ml, four of nine replicates were positive at 10(-1) PFU/ml, and zero of nine replicates were positive at 10(-2) PFU/ml). Concordant results were obtained in 24 of 28 clinical specimens after extraction by all methods. No evidence of contamination was observed after extraction by automated instruments. The data indicate that the sensitivity of enterovirus PCR is largely similar after extraction by QIAamp, BioRobot M48, and MagNA Pure; a trend of decreased sensitivity was observed after TRIzol extraction. However, the results of enterovirus PCR were largely concordant in patient samples, indicating that the four extraction methods are suitable for detection of enteroviruses in clinical specimens.     

QIAamp MinElute virus kit effectively extracts viral nucleic acids from cerebrospinal fluids and nasopharyngeal swabs.

BACKGROUND: Nucleic acid preparation from a variety of clinical specimens requires efficient target recovery and amplification inhibitor removal and is critical for successful molecular diagnosis. The QIAamp MinElute Virus kit (Qiagen Inc., Valencia, CA) was compared to the two existing methods currently used in our laboratory, IsoQuick (Orca Research Inc., Bothell, WA) for DNA extraction and RNAzol B (Leedo Laboratories Inc., Houston, TX) for RNA extraction, of viral nucleic acids. STUDY DESIGN: A total of 150 clinical specimens, including cerebrospinal fluid (CSF) and nasopharyngeal swabs (NPS), were used to determine the extraction efficiency of the MinElute compared to the other two methods. Nucleic acid recovery, hands-on time, turn-around-time and cost were compared across all kits. RESULTS: There was complete concordance between the MinElute and IsoQuick/RNAzol kits when herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), influenza A virus or enteroviruses were detected using a colorimetric microtiter plate PCR system. The kits were equivalent in their ability to detect either DNA or RNA with superior ability to recover a high quality and quantity of RNA. With the potential to process larger specimen volumes, the MinElute kit can significantly shorten processing time from 2h to 50-55min. CONCLUSIONS: Although relatively high test kit costs were noted, the MinElute kit provides another rapid and user-friendly specimen processing tool in the diagnostic molecular microbiology laboratory.     

Optimization of methods for detecting norovirus on various fruit

Methods for detecting norovirus (NoV) in food are crucial for investigation and prevention of outbreaks caused by NoV-contaminated food. However, current NoV detection methods have not been well examined or optimized. In this study, the effectiveness of various methods for eluting NoV from various fruit, concentrating the virus using polyethylene glycol (PEG), and extracting the viral RNA for subsequent assay by RT-PCR was optimized. First, six different buffers previously described for eluting NoV from fruit surfaces were evaluated. A known amount of NoV was spiked onto the surface of grapes, strawberries, and raspberries, and the virus was recovered with distilled water, 0.05 M glycine-0.14 M NaCl (pH 7.5), 2.9% tryptose phosphate broth-6% glycine, 100 mM Tris-HCl (pH 9.5), 50 mM glycine-50 mM MgCl(2) (pH 9.5), or 3% beef extract. Quantitation of the recovered virus using RT-PCR revealed that the most effective elution buffer was 3% beef extract. Secondly, to optimize a method for concentrating the recovered NoV, the key parameters of PEG precipitation, a typical method for concentrating enteric virus, were investigated. The influence of PEG molecular weight and the duration and temperature of the precipitation procedure were examined. NoV was concentrated most efficiently by precipitation when PEG (10,000) was used for 4h at room temperature. Finally, five different methods for nucleic acid extraction were evaluated. Among RNA extraction methods examined, QIAamp Viral RNA Mini kit showed the best recovery efficiency. Using the optimized method, approximately 6-80% of the seeded NoV was recovered from the various fruit.