保健食品中3种病原菌多重PCR快速检测方法的建立及验证

目的建立快速检测保健食品中沙门菌、志贺菌和金黄色葡萄球菌的多重PCR(multiplex PCR,m PCR)方法,并进行验证及初步应用。方法以提取的各菌株基因组DNA为模板,利用Gen Bank中登录的沙门菌inv A、志贺菌ipa H、金黄色葡萄球菌nuc基因序列设计引物,采用优化后的反应体系及确定的反应条件进行PCR扩增,扩增产物经3%琼脂糖凝胶电泳鉴定,对建立的方法进行特异性、敏感性、重复性验证。将沙门菌、志贺菌和金黄色葡萄球菌等量混合后,进行10倍系列稀释,加至10批无病原菌蛋白质粉和10批无病原菌减肥茶样品匀浆中,用建立的方法进行PCR扩增,并与GB常规生化检测结果进行比较。结果确定m PCR反应体系为:10×PCR buffer 2.0μl,d NTPs 2.5μl,Taq DNA酶0.6μl,上下游引物各1.5μl,Mg2+1.0μl,补加dd H2O至25μl。10株细菌中,沙门菌、2株志贺菌、金黄色葡萄球菌扩增结果为阳性,其他菌株为阴性,inv A、ipa H、nuc基因扩增片段与Gen Bank登录的序列同源性分别为99%、99%和100%;沙门菌、志贺菌和金黄色葡萄球菌菌株均能在101~105 CFU/ml浓度时扩增出特异性目的条带;5个浓度混合菌液扩增产物均可见特异性反应条带。该方法扩增出10批人工添加细菌蛋白质粉和10批人工添加细菌减肥茶中沙门菌、志贺菌和金黄色葡萄球菌的特异性基因片段,检出限均为102 CFU/ml,与GB常规生化检测结果一致。结论建立的保健食品中沙门菌、志贺菌和金黄色葡萄球菌m PCR检测方法特异性强,灵敏度高,重复性好,可满足不同检验机构快速、准确检测保健食品中多种病原微生物样品的实际需要。     

牛乳中蜡样芽孢杆菌荧光定量PCR检测方法的建立及验证

目的建立一种快速检测牛乳中蜡样芽孢杆菌的荧光定量PCR方法,并进行验证。方法根据GenBank中登录的蜡样芽孢杆菌gyrB基因序列及荧光定量PCR要求,设计合适的特异性引物,提取菌体DNA,对目的基因进行扩增及克隆;以重组质粒为模板进行荧光定量PCR扩增,绘制标准曲线;以蜡样芽孢杆菌DNA为模板进行荧光定量PCR扩增,确定其扩增条件。对建立的方法进行特异性及灵敏性验证。结果建立的方法检测其他3种牛乳中常见致病菌(大肠埃希菌、志贺菌、沙门菌)未见特异性扩增,即无交叉反应;最低检出限为1×10~(-7) ng/μL。以掺入蜡样芽孢杆菌的牛乳为样品,可检测到牛乳中10. 4 CFU/mL的蜡样芽孢杆菌,优于国标方法(GB 4789. 14-2014)检测含有相同菌落数的蜡样芽孢杆菌(仅能检测到1. 04×10~2 CFU/mL)。结论建立的方法特异性强,灵敏性高,适用于快速、准确检测牛乳中的蜡样芽孢杆菌,为实验室快速检测致病菌提供了参考。     

甲型肝炎病毒的RT-PCR-ELISA检测法

目的建立一种半定量检测甲型肝炎病毒(HAV)RNA的RT-PCR-ELISA方法。方法将5′端磷酸化的特异性探针包被在氨基化的微孔板上,以生物素标记的探针为引物,提取样品中的HAVRNA,反转录并扩增HAVDNA,变性后的PCR产物与探针杂交,并被捕获在微孔板上,再与酶标亲和素结合,显色测定。结果所建立的方法结果判断客观,灵敏度比凝胶电泳法高一个数量级,分析不同时间检测结果,差异无显著意义。结论已建立了半定量检测HAVRNA的RT-PCR-ELISA方法。     

皮内注射用卡介苗特异性鉴别试验多重PCR检测方法的建立及验证

目的建立皮内注射用卡介苗(Bacillus Calmette Guerin vaccine,BCG)特异性鉴别试验的多重PCR法,并进行验证。方法根据GenBank登录的Pasteur 1173P2株序列(AM408590. 1)设计并合成引物,以制备的BCG特异性鉴别试验国家参考品(简称BCG鉴别参考品)DNA为模板,多重PCR法扩增其特异性缺失区RD1,产物经3%琼脂糖凝胶电泳鉴定,验证方法的重复性、中间精密度、特异性、耐用性及灵敏度;采用该方法检测8批皮内注射用BCG供试品。结果 BCG鉴别参考品在重复检测6次、2名检测人员分别重复检测3 d、不同PCR退火温度及不同DNA聚合酶加量时均扩增出约200 bp的核酸片段;最低可检10 pg/mL的目的基因,仅对结核分枝杆菌H37Rv及皮内注射用BCG样品DNA扩增出特异性条带。经该方法检测,8批供试品PCR产物电泳均可见单一的目的条带,无RD1序列存在,大小与BCG鉴别参考品一致。结论多重PCR法的重复性、中间精密度、特异性、耐用性及灵敏度良好,可应用于皮内注射用BCG特异性鉴别试验。     

甲型肝炎病毒荧光定量RT-PCR检测方法的建立及在贝类检测中的应用

目的建立甲型肝炎病毒(hepatitis A,HAV)荧光定量逆转录PCR(qRT-PCR)检测方法,用于贝类中HAV的检测。方法选择HAV高度保守的5’UTR区设计引物及探针,以HAV质粒为标准品,HAV活病毒为样本,建立HAV qRT-PCR检测方法;验证该方法的特异性、灵敏度、重复性、准确性及中间精密度;采用建立的方法检测HAV活病毒和贝类样本,并与我国贝类HAV检测国标方法(GB/T22287-2008法)进行比较。结果通过14对引物探针分别对HAV质粒(101~108拷贝/μL)检测结果的比较,选择灵敏度最优的Pan1引物,建立了HAV qRT-PCR检测方法(PAN法)。PAN法对CVA16、CVB3、EVA71、PV等11种RNA病毒均无阳性扩增;灵敏度达10 TCID50/mL;检测2.699~5.699 LgTCID50/mL各浓度HAV活病毒的回收率为82.9%~127.0%,单人和双人检测的变异系数分别为3.0%~7.0%和2.6%~8.4%。PAN法检测HAV活病毒的灵敏度(10 TCID50/mL)优于GB/T22287-2008法;两种方法对20份贝类样本的检测结果均为阴性,符合率为100%。结论建立的PAN法具有良好的特异性、灵敏度、准确性、重复性和中间精密度,可用于贝类样本中HAV的核酸定量快速检测。     

大肠埃希菌O157:H7新型多重PCR检测方法的建立及其初步应用

目的建立一种能够满足现场快速检测、完全脱离实验室仪器设备的大肠埃希菌O157∶H7多重PCR检测方法。方法利用大肠埃希菌O157∶H7抗原基因保守序列fli Ch7和rfb E设计2对引物,并对Palm PCR G1-12退火温度、循环数、引物浓度及引物最佳比例进行优化,建立微量快速检测大肠埃希菌O157∶H7的多重PCR方法,并对该方法的特异性、灵敏性、重复性进行验证。取疑似病牛粪便共9份,利用粪便DNA提取试剂盒提取DNA,按照建立的PCR方法进行扩增,扩增产物经微型电泳仪电泳检测并照相。结果确定大肠埃希菌O157∶H7 PCR的最佳退火温度为54℃,循环数为25个循环,上下游引物浓度为10μmol/L,2对引物最适比为fli Ch7∶rfb E=1∶3。大肠埃希菌O157∶H7扩增产物可见625 bp(fli Ch7)和213 bp(rfb E)的特异性片段,而大肠埃希菌(ATCC25922)、猪链球菌2型、金黄葡萄球菌、鲍氏不动杆菌、小肠耶尔森菌、单增李斯特菌、沙门菌的PCR检测结果均为阴性,最低扩增DNA浓度为10 pg/μl,重复扩增5次均可见目的条带。9份疑似病牛粪便样品中,共检测出7份大肠埃希菌O157∶H7阳性样品。结论建立了大肠埃希菌O157∶H7微量快速多重PCR检测方法,该方法特异性、灵敏性和重复性均良好,可在现场约1 h报告结果,对突发性传染病的防治具有重要意义。     

聚合酶链式反应技术检测化妆品中的铜绿假单胞菌

利用聚合酶链式反应(PCR)技术建立了一种快速检测化妆品中铜绿假单胞菌的方法。根据已报道的铜绿假单胞菌特异性基因ETA设计引物,确定引物的特异性和灵敏度;对染菌化妆品样品进行增菌检测。琼脂糖凝胶电泳检测PCR产物结果显示,仅在含有铜绿假单胞菌基因组的样品中得到条带;对染菌样品的扩增结果显示,可以在原样品的活菌浓度为6 cfu.mL-1时通过增菌12 h后检出。     

金黄葡萄球菌核酸探针检测方法的建立

目的建立快速检测食品中金黄葡萄球菌的核酸探针检测法。方法通过使用吖啶酯标记的特异DNA探针检测金黄葡萄球菌,确定最高和最低探针浓度,验证该方法的特异性及敏感性,并与传统国标法的检测结果进行比较。结果核酸探针法最高探针浓度为2pmol∕50μl,最低探针浓度为0.25pmol∕50μl;该方法特异性良好,检出纯培养菌落最低限约为106cfu∕ml;与国标法检测结果一致性较高。结论核酸探针法可用于食品中金黄葡萄球菌的快速检测。     

双重荧光定量PCR技术在药品微生物检验中的应用

目的建立快速检测金黄色葡萄球菌和铜绿假单胞菌的双重荧光定量PCR方法并在药品微生物检测中应用。方法通过设计特异性引物和探针,扩增金黄色葡萄球菌的femB基因和铜绿假单胞菌的DNA gyrase subunit B基因,建立荧光定量PCR方法。将该方法应用于30批次药品微生物检测。结果建立同时检测金黄色葡萄球菌和铜绿假单胞菌的双重荧光定量PCR方法,从DNA提取到检测完毕仅需2 h。检测灵敏度为50 copies/μL,特异性为100%。30批次样品检验结果表明该方法与传统细菌培养方法结果一致。结论该法缩短了检测时间,具有良好的灵敏性和特异性,在药品微生物检验中有很好的应用前景。     

小体积水样中鼠伤寒沙门菌氨基磁珠富集的LAMP可视化检测方法

研究建立了小体积(1 mL)水样中鼠伤寒沙门菌氨基磁珠富集-环介导等温扩增(LAMP)可视化检测方法。研究对氨基磁珠富集水中鼠伤寒沙门菌的pH和时间进行优化，考察了氨基磁珠富集-LAMP可视化检测方法的特异性、抗干扰能力和灵敏度，并用所建立的方法对人工模拟水样和实际水样进行检测。在pH值=7.0的条件下，氨基磁珠与水样作用60 min,氨基磁珠对水中的鼠伤寒沙门菌的捕获效率接近100%。氨基磁珠富集-LAMP扩增体系特异性好，抗干扰能力强，对水中鼠伤寒沙门菌的检测灵敏度为2.39 CFU/mL,阳性结果可直接观察到反应管由橙黄色变成草绿色，而阴性体系不变色。将所建立的方法用于人工模拟水样，检测结果均为阳性，对成都市新都区河流或景观湖等水样检测，共检出1份鼠伤寒沙门菌阳性水样。本研究建立的氨基磁珠富集-LAMP方法能够实现小体积水样中鼠伤寒沙门菌高效、准确的可视化检测。     

Interactions between shrinkage reducing admixtures (SRA) and cement paste's pore solution

Shrinkage reducing admixtures (SRA) have been developed to combat shrinkage cracking in concrete elements. While SRA has been shown to have significant benefits in reducing the magnitude of drying and autogenous shrinkage, it has been reported that SRA may cause a negative side effect as it reduces the rate of cement hydration and strength development in concrete. To examine the influence of SRA on cement hydration, this study explores the interactions between SRA and cement paste's pore solution. It is described that SRA is mainly composed of amphiphilic (i.e., surfactant) molecules that when added to an aqueous solution, accumulate at the solution-air interface and can significantly reduce the interfacial tension. However, these surfactants can also self-aggregate in the bulk solution (i.e., micellation) and this may limit the surface tension reduction capacity of SRA. In synthetic pore solutions, SRA is observed to form an oil-water-surfactant emulsion that may or may not be stable. Specifically, at concentrations above a critical threshold, the mixture of SRA and pore fluid is unstable and can separate into two distinct phases (an SRA-rich phase and an SRA-dilute phase). Further, chemical analysis of extracted pore solutions shows that addition of SRA to the mixing water depresses the dissolution of alkalis in the pore fluid. This results in a pore fluid with lower alkalinity which causes a reduction in the rate of cement hydration. This may explain why concrete containing SRA shows a delayed setting and a slower strength development.     

Influence of bleed water reabsorption on cement paste autogenous deformation

The effects of bleed water reabsorption and subsequent early age expansion on observed autogenous deformation are investigated in this research. Bleeding was induced by varying superplasticizer and shrinkage-reducing admixture dosages and by increasing the water-to-cement ratio. This research revealed that significant early age expansion occurs with increasing chemical admixture dosages and higher water-to-cement ratios, as expected, due to increasing bleeding of those samples. When samples were rotated, negligible early age expansion was observed. Thus, bleed water reabsorption is shown to be the primary mechanism causing initial expansion in sealed autogenous deformation samples. Thermal dilation and ettringite growth appear to have a minimal influence on the observed expansion. Rotating the samples during setting eliminates the potential for bleed water reabsorption and is recommended for all autogenous deformation testing.     

Chemical model for cement-based materials: Thermodynamic data assessment for phases other than C-S-H

In the context of waste confinement, concrete may be used both as a confinement and as a building material. Concerning radwaste, the heat released during radioactive decay will modify the equilibrium constants of the minerals forming the concrete. The present work aims to elucidate the temperature dependency of the thermodynamic functions related to minerals from the concrete or associated with some of its degradation products. A large set of experimental data has been collected, for the chemical systems SO₃-Al₂O₃-CaO-CO₂-Cl-H₂O and SiO₂-Al₂O₃-CaO-H₂O, including iron and magnesium bearing phases. Most of the data collected concern experiments in aqueous media but results from calorimetric studies were also included, when available. Based on selected thermodynamic properties for each phase, predominance diagrams were drawn for the chemical elements listed above. Phase relations reported into predominance diagram appear rather consistent with most of the literature results. The case of katoite has been especially discussed, because it shows inconsistencies with respect to a hydrogarnet-grossular solid solution and with respect to phase relations reported into already published works. Finally, we underline the chemical compatibility of Portland cement pastes with carbonate aggregates, compared to silicates, for long-term storage applications.     

An investigation of microstructure evolution in cement paste through setting using ultrasonic and rheological measurements

The response of hydrating cement paste through setting are monitored using rheological measurements and ultrasonic reflection measurements. Increases in the elastic modulus and yield stress of cement paste with time are obtained from the rheological measurements. Ultrasonic measurements are performed using horizontally polarized shear waves (SH) reflected off of the hydrating cement paste. Changes in the ultrasonic signal through setting are related with changes in the porosity and stiffness of an equivalent water-filled poroelastic material, which provides identical acoustic impedance. The measured changes in the shear modulus obtained from ultrasonic measurement are shown to correlate well with increase in elastic modulus obtained from rheological measurements. The increase in the shear modulus of the porous material obtained from the ultrasonic measurements is shown to correspond well with the observed increase in the yield stress of the cement paste. By combining the information from rheological and ultrasonic measurements, it is found that even in the fluid stage there is sufficient structural integrity in the arrangement of cement grains to support low-amplitude shear stress and the evolution of a continuously connected network of cement particles within the paste is coincident with a rapid increase in the shear modulus of the porous skeleton.     

Nonlinear ultrasonic evaluation of load effects on discontinuities in concrete

The presence of discontinuity surfaces in concrete structures, i.e. two or more layers in contact, may be an existing situation with evident relapses on damage formation and progression. Differences occur depending on the type of discontinuity, which could be a thin weaker layer or a pre-existing crack. The behavior of pre-existing interfaces is here studied by means of the Scaling Subtraction Method, a Nonlinear Ultrasonic Non-Destructive Technique, that revealed to be effective in describing the mechanical evolution of concrete samples with discontinuity surfaces under the effects of compressive loads.     

Assessment of the long-term stability of cementitious barriers of radioactive waste repositories by using digital-image-based microstructure generation and reactive transport modelling

Cement-based grout plays a significant role in the design and performance of nuclear waste repositories: used correctly, it can enhance their safety. However, the high water-to-binder ratios, which are required to meet the desired workability and injection ability at early age, lead to high porosity that may affect the durability of this material and undermine its long-term geochemical performance.In this paper, a new methodology is presented in order to help the process of mix design which best meets the compromise between these two conflicting requirements. It involves the combined use of the computer programs CEMHYD3D for the generation of digital-image-based microstructures and CrunchFlow, for the reactive transport calculations affecting the materials so simulated. This approach is exemplified with two grout types, namely, the so-called Standard mix 5/5, used in the upper parts of the structure, and the “low-pH” P308B, to be injected at higher depths.The results of the digital reconstruction of the mineralogical composition of the hardened paste are entirely logical, as the microstructures display high degrees of hydration, large porosities and low or nil contents of aluminium compounds.Diffusion of solutes in the pore solution was considered to be the dominant transport process. A single scenario was studied for both mix designs and their performances were compared. The reactive transport model adequately reproduces the process of decalcification of the C-S-H and the precipitation of calcite, which is corroborated by empirical observations. It was found that the evolution of the deterioration process is sensitive to the chemical composition of groundwater, its effects being more severe when grout is set under continuous exposure to poorly mineralized groundwater. Results obtained appear to indicate that a correct conceptualization of the problem was accomplished and support the assumption that, in absence of more reliable empirical data, it might constitute a useful tool to estimate the durability of cement-based structures.     

Wavelet-based analysis of ultrasonic longitudinal and transverse pulses in cement-based materials

Ultrasonic pulse velocity has been used for decades to detect localized damage and to estimate concrete properties. More recent applications aim at diffuse damage characterization, such as environmental and mechanical damage. In most applications the methodology to calculate pulse speed is a very important issue. This work, applying continuous wavelet transform (CWT) to construct time-frequency signal representations, calculates frequency-dependent velocity of longitudinal and transverse ultrasonic pulses using wavelet scales. The method is applied to a total of 14 specimens of 5 different mixes and frequency-dependent velocities are calculated using four wavelet families. The CWT capability to decompose the inquiring pulse spectrum and analyze phase velocities is discussed with regard to wavelet, pulse type, and mixture. Frequency-dependent velocity of longitudinal pulses at lower frequencies (from 100 kHz up to 250 kHz) was proven to be much more sensitive to mix proportions than transverse pulses.     

Effects of adhesive type and polystyrene concentration on the shear strength of bonded polystyrene/high-density polyethylene blends

The lap-shear strengths of adhesively bonded polystyrene (PS), high-density polyethylene (HDPE), and their blends, were studied as a function of adhesive type and blend composition. The performance of virgin and recycled polymer systems was examined. The lap-shear strength depended strongly on the amount of PS in the blend and the type of adhesive, and the acrylic adhesives demonstrated the best performance for all compositions. Bonded strengths of HDPE increased by approximately 50% when HDPE was blended with 34% PS, the co-continuous composition. The results indicate that structural elements made from PS/HDPE immiscible blends may be effectively bonded with adhesives without expensive surface treatments.