基于刃天青的电化学方波伏安法快速鉴别大肠杆菌的耐药性

目的：评价5株食源性大肠杆菌临床分离株对环丙沙星、庆大霉素和氨苄西林的抗生素耐药性。方法：基于细菌能量代谢过程中的电子传递理论,引入刃天青作为氧化还原探针,建立了一种基于刃天青介导的大肠杆菌耐药性快速鉴别的电化学方波伏安法（square wave voltammetry,SWV）。结果：在1.69×10²～1.69×10⁸ CFU/mL范围内,大肠杆菌浓度与刃天青氧化峰电流（I）之间存在较好的线性关系,回归方程为Y=-0.156 0X-2.965 5,R²=0.977 5;在进行大肠杆菌耐药性测定时,参考美国临床和实验室标准协会制定的药敏试验标准,以刃天青的氧化峰电流（I₀）和大肠杆菌直接与刃天青作用后得到的氧化峰电流（I_-a）作为对照,测定大肠杆菌用抗生素处理后与刃天青作用得到氧化峰电流（I_+a）,引入细菌活性值V_ETA作为电化学方波伏安法鉴别细菌耐药性的指标,建立了刃天青介导的大肠杆菌电化学快速药敏试验方法;运用建立的方法检测了5种食源性大肠杆菌临床分离株对3种抗生素的耐药性,以V_ETA=50作为判断细菌敏感和耐药的临界值,均得到与纸片扩散法结果相符的药敏试验结果,而纸片扩散法报告结果需18 h,该法仅需2 h。结论：建立的基于刃天青介导的大肠杆菌耐药性快速鉴别的电化学方波伏安法能够准确且快速检测5株食源性大肠杆菌临床分离株对环丙沙星、庆大霉素和氨苄西林的抗生素耐药性。     

灭菌乳中嗜热脂肪芽孢杆菌商业无菌的最低可视浓度的测定

目的 测定灭菌乳中嗜热脂肪芽孢杆菌的商业无菌检出限。方法 随机购买3种不同品牌的灭菌乳,分别添加10_⁵、10_⁴、10_³、10_²、10_¹、10_⁰ CFU/mL浓度的嗜热脂肪芽孢杆菌制成模拟样品,分别对培养4 d、7 d、9 d、10 d的样品按照GB 4789.26-2013《食品安全国家标准 食品微生物学检验 商业无菌检验》的检验方法检测。结果 3种品牌牛乳的模拟样品商业无菌的最低检出浓度均为10_¹ CFU/mL,同时发现培养10 d后样品中的嗜热脂肪芽孢杆菌并无明显的增殖也无菌落进入休眠状态。对培养10 d后的模拟样品进行菌落计数验证以上结论。结论 灭菌乳中嗜热脂肪芽孢杆菌商业无菌检出限为10_¹ CFU/mL,如果灭菌乳中存在嗜热脂肪芽孢杆菌浓度＜10_¹ CFU/mL时,可能在检验过程中对灭菌乳的芽孢菌出现漏检。灭菌乳中嗜热脂肪芽孢杆菌在培养后无明显增殖,符合商业无菌相对无菌标准。     

分散固相萃取-液相色谱-串联质谱法测定植源性油料油脂中67种除草剂

目的 建立分散固相萃取结合液相色谱-串联质谱同时测定油料油脂中67种除草剂残留的检测方法。方法 选取花生、大豆、油菜籽、花生油、大豆油及菜籽油为典型基质试样,经1%甲酸-乙腈提取,通过乙二胺基-N-丙基和无水MgSO₄分散固相萃取净化。以甲醇和5 mmol/L乙酸铵（含0.1%甲酸）为流动相梯度洗脱,采用C₁₈色谱柱分离,ESI离子源正负离子模式同时进行多反应监测（MRM）,基质标准曲线外标法定量。结果 基质加标试验结果表明,0.000 5~0.08 mg/L浓度范围内67种除草剂线性良好,决定系数（R²）均>0.992,定量限（LOD）为0.005~0.02 mg/kg。3个加标水平下（LOD、2LOD、10LOD）的回收率为62.3%~118.1%,RSD（n=6）均<15%。结论 本方法稳定性高、特异性强、灵敏、准确、高效,可以用于油料油脂中多种除草剂残留的定性定量检测。     

纳米Ag@TiO2、Fe@TiO2和Zn@TiO2的抑菌活性比较研究

探索常见金属离子掺杂对抑菌片的抑菌性能的影响,为新型纳米TiO₂抑菌片的制备与改善提供技术基础。选择固定质量分数的Ag⁺、Zn²⁺和Fe³⁺掺杂TiO₂,通过抑菌圈、MIC与MBC方法综合评价其抑菌性能,对微观结构进行表征分析,探讨抑菌性能差异的原因。结果表明:抑菌性能大小顺序为Ag@TiO₂> Zn@TiO₂> Fe@TiO₂,Zn@TiO2 的抑菌活性较好但抑菌稳定性差Ag@TiO2对大肠杆菌和金黄色葡萄球菌的MIC、MBC均是10、20 mg/L,Zn@TiO₂对大肠杆菌和金黄色葡萄球菌的MIC、MBC均是30、50 mg/L。表征发现抑菌活性强弱主要归因于纳米TiO₂表面与金属离子共同产生的活性位点数量及稳定性,与孔结构或表面宫能团的关联不大。     

婴幼儿配方乳粉和发酵乳中沙门氏菌检验方法的比对研究

目的 对比美国FDA/BAM、ISO 6579-2017和GB4789.4-2016沙门氏菌检验方法的检出限、灵敏度,筛选出针对婴幼儿配方乳粉、发酵乳产品以及环境样品中沙门氏菌污染最灵敏的检测方法。方法 对20种血清型沙门氏菌进行平行检测,对比美国FDA/BAM、ISO 6579-2017和GB4789.4-2016沙门氏菌检验方法的检出限、灵敏度。使用不同基质(婴幼儿配方奶粉、酸奶样品和环境样品)人工染菌样品,对三个方法的样品适用性进行比较与评价。结果 三个标准方法的检出限为10_^-1-10_⁰CFU/样品。FDA/BAM方法、ISO 6579-2017方法、GB 4789.4-2016方法在10_¹CFU/样品染菌水平灵敏度均为100%;10_⁰CFU/样品染菌水平,灵敏度为95%、90%、90%;10_^-1CFU/样品染菌水平,灵敏度为65%、50%、45%。对三类人工污染沙门菌的样品,FDA/BAM方法的检出率显著高于ISO和GB4789方法(P＜0.05),ISO和GB4789方法检出率无显著性差异(P>0.05)。结论 三种方法检出限一致 ,均可以有效检测食品中沙门氏菌。染菌浓度低于10_⁰CFU/样品时, FDA/BAM方法的灵敏度显著高于ISO和GB4789方法。不同基质人工污染沙门氏菌样品对比,FDA/BAM方法的检出率显著高于ISO6579-2017方法和GB4789.4-2016方法。     

基于HAND系统5种致腹泻大肠杆菌多重实时PCR初筛方法的建立

以实验室17株种属关系较近的菌株进行特异性评价,通过溶解曲线Tm值对扩增产物进行分析表明,该方法具有高特异性,可同时检出5种致泻性大肠杆菌EAEC(aggR)、EHEC/EPEC (eae)、ETEC (LT)和EIEC(ipaH)的相关毒力基因。粪便模拟样本试验表明,其敏感性达10~4～10~6 CFU/mL。建立的多重实时PCR检测方法适用于5种致泻性大肠杆菌的初步筛选。     

食品检测用产肠毒素大肠埃希氏菌标准物质的制备及评价

目的 制备并评价产肠毒素大肠埃希氏菌（ETEC）标准物质。方法 分别用基质辅助激光解吸电离（MALDI-TOF MS）、生化、16 s RNA基因序列测定三种方法确认菌株种属。采用冷冻干燥技术制备600个ETEC标准样品（10³ CFU/样品）;从中随机抽取20个进行均匀性检验;模拟25 ℃和37 ℃的运输温度测试样品的运输稳定性,同时在4 ℃和-20 ℃的条件下进行保藏稳定性检验。组织 3家实验室对样品进行协同验证。最后选择20件食品基质来验证样品的使用效果。结果 生化、MALDI-TOF MS、16 s RNA基因序列鉴定CMCC（B）43208为大肠埃希氏菌, lt、stp、sth基因确认其为ETEC。均匀性检验中,单因素方差分析得F=1.48,小于F_INV（0.05,19,20）。稳定性检验中,在25 ℃和37 ℃保藏7 d后结果为10³ CFU/样品,在-20 ℃保藏60 d和4 ℃保存28 d后结果为10³ CFU/样品。协同标定实验中,3 家实验室检测样品中菌株均为ETEC（10³ CFU/标准样品）。使用效果评价中,ETEC标准样品加入20种食品基质后均可检出,而本底对照均未检出。结论 本实验制备的ETEC标准样品均匀且稳定,协同验证、食品基质验证实验结果均与预期设置的10³ CFU/样品一致,可以作为标准物质使用。     

微咸水灌溉条件下施用不同改良剂对盐渍化土壤盐分离子分布的影响--测试文章

利用5种不同的土壤改良剂,对矿化度在2～3 g·L^-1的微咸水灌溉棉田土壤进行改良效果研究。结果表明：五种改良剂均降低土壤pH值和总盐含量,并能有效控制土体Na⁺、Ca²⁺、SO₄^2-、HCO₃^-积累;其中,磷石膏能显著降低土体Na⁺、Ca²⁺、SO₄^2-总含量（P＜0.05）,DS1997能显著降低土体Na⁺、HCO₃^-总含量（P＜0.05）,酸碱平衡剂显著降低土体Ca²⁺、SO₄^2-总含量（P＜0.05）,禾康改良剂有效控制土体SO₄^2-、HCO₃^-含量;改良剂对土体中Cl^-改良效果不显著（P＞0.05）。研究得出：微咸水灌溉导致土壤pH值升高和含盐量增加,造成土壤盐分的积累;土壤改良剂可有效减少微咸水灌溉引起的盐分积累,改善土壤理化特性和盐分离子分布。     

柠檬明串珠菌KM20中D-乳酸脱氢酶的特性

目的：分析柠檬明串珠菌中D-乳酸脱氢酶（D-LDH）的酶学特性。方法：对柠檬明串珠菌KM20中D-乳酸脱氢酶基因进行克隆表达并构建表达质粒,转化至Escherichia coli BL21（DE3）中实现过表达。结果：经Ni-NTA柱亲和层析纯化后,D-LDH-1与D-LDH-2编码的蛋白分子质量分别为40.0,38.5 kDa;比活力分别为2.18,153.10 U/mg;在丙酮酸还原中两种酶的最适pH值与最适温度均为8.0与40 ℃;而乳酸氧化时D-LDH-2的最适pH值与最适温度分别为12.0与30 ℃。D-LDH-1与D-LDH-2对草酰乙酸、苯丙酮酸和2-酮戊二酸具有较强的催化能力,且Ca²⁺、Cu²⁺和Na⁺对其酶活性均具有促进作用,Zn²⁺与SDS对酶活性有极高的抑制作用。此外,两种酶对丙酮酸的K_m值分别为2.98,6.11 mmol/L,对丙酮酸的K_cat/K_m分别为6.04×10²,2.28×10⁴ L/（mol·s）,LDH-2对D-乳酸的K_cat/K_m为65.0 L/（mol·s）。结论：D-LDH-1与D-LDH-2为柠檬酸明串珠菌中催化D-乳酸合成的关键酶。     

微波功率对月柿果片微波间歇干燥中水分迁移及品质的影响

目的：探讨微波间歇干燥对月柿果片水分动态和微观结构的影响。方法：选取不同微波功率（280,350,420,490,560 W）对月柿果片进行微波间歇干燥,对干燥过程中月柿果片的水分动态和迁移进行监测,以及对月柿果片的色泽、质构、微观结构进行测定分析。结果：新鲜月柿中的水分主要以结合水（T₂₁）、不易移动水（T₂₂）和自由水（T₂₃）3种形式存在,分别对应3个明显的水峰。随着干燥时间的延长,不易移动水和自由水的横向弛豫时间显著降低。L^*、a^*、b^*值随着微波功率的增加显著减小,ΔE、硬度、弹性、咀嚼性随着微波功率的增加而增加。随着微波功率的增加,果片的组织结构收缩塌陷越明显。结论：较低的微波功率能得到品质较佳的月柿干制品。     

Rapid Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Food Using a Recombinase Polymerase Amplification-Based Assay

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 10² copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.     

Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR

The objective of this study was to develop a rapid multiplex PCR (m-PCR) method using pure bacterial cultures and pork that would allow the simultaneous detection of five major foodborne pathogens likely to be found in pork (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella and Yersinia enterocolitica). Five pairs of primers were designed to identify invA gene, hlyA gene, rfbE gene, nuc gene and ail gene for Salmonella, L. monocytogenes, E. coli O157:H7, S. aureus and Y. enterocolitica, respectively. On the basis of determining specificity of the m-PCR, sensitivity was performed with an overnight enrichment step in culture media and pork. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The results suggested that the m-PCR was an effective procedure having high specificity for the simultaneous detection of the five target pathogens. After overnight culture, the detection limit was 10³ cfu/mL for the simultaneous detection of the five target pathogens and less than 10 cfu/mL for detection of a single pathogen. The m-PCR protocol successfully detected all five organisms inoculated overnight together on pork. Salmonella, S. aureus, L. monocytogenes, Y. enterocolitca and E. coli O157:H7 were detected at levels of 142, 51, 9, 33 and 670 cfu/mL, respectively, in the pork. The m-PCR assay developed in this study could provide an effective and informative supplement for routine monitoring for pork safety.     

Saltatory rolling circle amplification for sensitive visual detection of Staphylococcus aureus in milk

Monitoring Staphylococcus aureus with high sensitivity is very important for ensuring milk quality and food safety. In this study, we used a rapid nucleic acid isothermal amplification method, saltatory rolling circle amplification (SRCA), for the detection of Staph. aureus in milk. The results of the SRCA method can be assessed visually by the presence of white precipitate or by fluorescence measurement. Thirteen Staph. aureus strains and 31 non-Staph. aureus strains were used to evaluate the specificity of SRCA. The method exhibited excellent detection of Staph. aureus genomic DNA at a concentration of 7.8 × 10¹ fg/µL when assessed by visible precipitate, and at 7.8 × 10⁰ fg/µL when detected by fluorescence after addition of the fluorochrome SYBR Green I. In artificially inoculated milk, the detection limits of SRCA were 5.6 × 10² cfu/mL by precipitate and 5.6 × 10¹ cfu/mL by fluorescence, respectively. Compared with conventional PCR approaches, the SRCA assay achieved at least 100-fold higher sensitivity. Moreover, the sensitivity, specificity, and accuracy of the SRCA-based system were calculated to be 100.00, 97.73, and 97.78%, respectively. These results indicate that SRCA has potential application as a sensitive and visual technique for the detection of Staph. aureus in milk.     

Development and application of a sensitive,rapid, and reliable immunomagnetic separation-PCR detection method for Cronobacter spp.

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 10² cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 10² cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 10¹ cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods.     

Identification and quantification of acetic acid bacteria in wine and vinegar by TaqMan–MGB probes

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10²–10³ cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S–23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA.     

Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10⁰ cfu/mL and 5.7 × 10¹ cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.     

环介导等温扩增方法在食品中沙门菌检测的应用和评价

将环介导等温扩增检测方法应用于食品中沙门菌的检验,并在检测方法特异性、灵敏度等方面与实时荧光PCR和传统检测方法进行比较。方法 针对沙门菌属高度保守的fimY基因设计环介导等温扩增检测引物并优化反应体系,在特异性、灵敏度和实际样品检测等方面与实时荧光PCR及传统检测方法比对。结果 本研究建立的LAMP方法检测沙门菌93株和非目标菌31株,具有良好的特异性。在纯培养、无需增菌情况下,其检测灵敏度为6.4×10²cfu/ml,与实时荧光PCR方法相当。食品基质添加试验中,环介导等温扩增方法检测低限为2cfu/25g样品;对45份实际食品样品检测结果表明,该方法实际样品检出率为11.1%,与实时荧光PCR及传统方法检测结果一致。结论 本研究建立的沙门菌环介导等温扩增检测方法具有良好的特异性,检测灵敏度与实时荧光PCR相当,适用于沙门菌的快速筛选。     

QCM immunosensor with nanoparticle amplification for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) immunosensor was developed and evaluated for detection of Escherichia coli O157:H7. The immunosensor was fabricated by self-assembling of protein A and affinity-purified anti-E. coli O157:H7 antibodies on the gold electrode of an AT-cut piezoelectric quartz crystal. To enhance the sensitivity of the QCM immunosensor, nanoparticle-antibody conjugates, which were prepared using streptavidin-conjugated nanoparticles (145 nm diameter) and biotinylated anti-E. coli antibodies, were used for signal amplification. After the binding of E. coli O157:H7 cells with the antibodies immobilized on the electrode, nanoparticle-antibody conjugates were introduced as mass amplifiers. Compared to the direct detection of E. coli O157:H7, the binding of the nanoparticle conjugates further resulted in a decrease in resonant frequency and an increase in resonant resistance, and the detection sensitivity was improved by five orders of magnitude by lowering the detection limit from 10⁷ to 10² CFU/mL. The sensor specificity and nonspecific adsorption of nanoparticle-antibody conjugates were also investigated.     

Simultaneous determination of five quinoxaline-1,4-dioxides in animal feeds using an immunochromatographic strip

An immunochromatographic (ICG) strip was developed for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides in animal feed. For this purpose, polyclonal antibodies (PcAb) with group-specific quinoxaline-1,4-dioxides were conjugated to colloidal gold particles as the detection reagent for ICG strips to test for quinoxaline-1,4-dioxides. This method achieved semi-quantitative detection of quinoxaline-1,4-dioxides within 5–10 min. The visual lower detection limits of the strip for quinocetone, cyadox, carbadox, mequindox and olaquindox were 10, 15, 15, 20 and 20 ng ml^?1, respectively. Using an ICG strip reader, the 50% inhibitions (IC₅₀ values) were calculated to be 9.1, 13.5, 16.6, 20.2 and 21.3 ng ml^?1 for quinocetone, cyadox, carbadox, mequindox and olaquindox, respectively. When used to analyse samples of animal feed, acceptable recovery rates of 77.5–99.5% and coefficients of variation (CVs) of 4.3–10.7% were obtained. Levels measured with the ICG strip for 10 spiked samples were confirmed by HPLC with a high correlation coefficient of 0.9965 (n = 10). In conclusion, the method was rapid and accurate for simultaneous determination of five quinoxaline-1,4-dioxides antibiotics in animal feed.