基于ATP生物发光法的微生物数量快速检测技术的研究进展

微生物数量的快速检测一直是工业生产与食品行业需要解决的问题,腺嘌呤核苷三磷酸(adenosine triphosphate,ATP)生物发光法具有操作简便、检测周期短等优点,可满足一般微生物检测的需求。然而,ATP生物发光法的准确性也受到不同因素的影响,如微生物的ATP检测限值较高、微生物自身及其他因素(如非微生物ATP、提取剂种类、荧光素酶活性等)均对微生物数量的检测产生影响。本文简述了不同微生物数量检测方法的优缺点,介绍了ATP生物发光法的发展历程及原理,综述了非微生物ATP与游离ATP、微生物量、ATP提取剂、荧光素酶等因素对ATP生物发光法灵敏度与稳定性的影响,归纳总结了ATP生物发光法及检测设备在食品、医疗、污水处理等领域的应用现状,并就ATP生物发光法体系的优化及ATP在线检测的应用等方面进行了展望,以期为ATP生物发光法的高效应用提供新的思路。     

ATP的生物发光与清洁

洗手、刷牙,清除污物、杀灭病菌,看似常规小事,其实不然。它与预防疾病、增进身体健康,保障食品、药品、半导体元件、航天器材等相关工业制品的安全品质,提升医院、餐馆、托儿所等各类公共场所服务水平有着密切关系。一句话：清洁、消毒是件关乎百姓福祉以及和谐、文明社会建设的重要窦事。     

生物发光法微生物快速检测试剂的性质及其影响因素研究

研究了ATP生物发光微生物快速检测试剂的反应动力学、反应最适温度、pH以及各种影响因素。在ATP生物发光反应中,测试系统中D-荧光素用量为40～50μg／mL时对反应已经足够;发光脉冲计数CPM值随反应时间的延长不断降低,开始的1min内,其CPM值下降最快,然后下降速度不断减缓;反应的最适温度为24℃～25℃;而体系的最佳pH为7．2-7．4。配制好的发光试剂溶液置于4℃保存45h,可以保持86％的活力。在25℃时保温1h,活力下降较少,随时间的增加,活力逐渐下降,到6．5h时,仅剩53．5％的活力,而在33℃时,随着保温时间的延长,酶活力下降较快,保温1．5h,活力剩下59．1％,因此保存温度对发光试剂活力的影响非常大。各种化学物质如酸、碱、盐及表面活性剂都会抑制ATP发光反应,当NaCl浓度达到1．5g／L时,即可以抑制52．5％的发光,TritonX-100及酸、碱对系统均有一定的影响,CTAB、SDS及TCA则严重抑制发光反应。     

基于重组溶葡球菌酶和ATP生物发光技术特异定量检测金黄色葡萄球菌

基于重组溶葡球菌酶和ATP生物发光法建立特异定量检测金黄色葡萄球菌的方法。优化设计合成溶葡球菌酶序列,构建重组表达载体pQE30-Lys,转化至大肠杆菌M15并诱导表达,镍柱纯化得到目的蛋白。利用重组溶葡球菌酶和ATP生物发光法特异定量检测金黄色葡萄球菌并与平板计数对比。成功表达了重组溶葡球菌酶,并建立了特异定量检测金黄色葡萄球菌的方法,与平板计数具有显著线性关系。本研究建立的将重组溶葡球菌酶和ATP生物发光法相结合的检测方法操作快捷简单,具有良好的应用前景。     

扩增内标及其在食源性致病菌PCR检测中的应用

虽然PCR技术不断地得到了发展和改进,但检测结果容易出现假阴性而影响检测准确性的现象一直没有得到很好的解决。现在大多数学者普遍认为,在PCR体系中加入扩增内标(即一段人工构建合成的DNA序列或者是一段致病菌的看家基因序列)能有效指示假阴性现象的出现,是PCR检测技术标准化的措施之一。本文将从PCR检测方法中假阴性出现的原因、扩增内标的构建以及扩增内标在PCR检测方面的应用三方面进行综合评述,并结合本实验室的工作基础,介绍扩增内标的简捷构建过程和应用要点,希望在不影响检测灵敏度的前提下,发挥扩增内标对假阴性的指示作用。     

基于裂解酶与ATP生物发光法特异定量检测炭疽杆菌方法的研究简

目的：原核表达炭疽杆菌噬菌体叮裂解酶（PlyG）和萤光素酶（Luc）,结合这2种酶建立特异定量检测炭疽杆菌的方法。方法：PCR扩增得到带有His标签的裂解酶基因和萤光素酶基因,构建重组表达载体pET22b-p@G和DET22b-luc,转化至大肠杆菌BL21（DE3）并诱导表达,过镍柱纯化得到目的蛋白;利用裂解酶裂解和ATP生物发光定量检测蜡样芽孢杆菌RSVFl,与平板计数法对比建立线性关系。结果：表达了炭疽杆菌噬菌体γ裂解酶和萤光素酶,并建立了特异定量检测蜡样芽孢杆菌RSVF1的方法,与平板计数方法具有显著的线性相关。结论：因炭疽杆菌与蜡样芽孢杆菌RSVF1均对PlyG具有较强的敏感性,故本研究所建立的将炭疽杆菌噬菌体γ裂解酶与萤光素一萤光素酶系统相结合的检测方法对现场或临床定性定量检测炭疽杆菌提供了理论支持,具有良好的应用前景。     

ATP的生物发光与清洁

北京人居环境生态条件已有明显改善,但若干中小型食品生产加工单位饮食摊点、公共服务部门场所设施的微观清洁卫生水平离"世界城市"要求还有很大差距。发达国家(欧盟、北美、日本等)均已将ATP生物发光检测方法列入相关卫生标准检测方法并建立与其对应的卫生"评价体系"和"卫生基准值制度"。     

添加有扩增内标的副溶血弧菌PCR检测方法的建立

摘要：【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌（Vibrio parahaemolyticus）基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物（vp1332L/vp1332R）,同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343 bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499 bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102 cfu/mL。人工污染实验表明,起始染菌量为1.24 cfu/25 g样品时经8 h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

一种利用Profile-1 生物发光仪快速测定土壤中微生物量的改良方法

开发了一种利用Profile-1生物发光仪测定土壤中微生物量的改良方法,并以此方法分别测定了标准大肠杆菌茵液以及3种不同类型的土壤(九段沙湿地土壤,崇明东滩大田土壤和崇明实验地改良土壤)的微生物量,并将结果与Profile-1生物发光仪自带的标准分析方法以及传统的菌落计数法进行比较。结果显示,改良的ATP提取方法(BAB改良分析法)和Profile-1生物发光仪自带的标准分析方法都可用于液体样品中微生物量的测定,其灵敏度和准确度无显著差异(P0.05)。但在测定土壤样品时,菌落计数法测定结果大约占BAB改良分析法测定结果的1%～5%,占Profile-1生物发光仪自带的标准分析方法的测定结果的22%～99%。这表明在分析土壤样品时,BAB改良分析法较Profile-1生物发光仪自带的标准分析方法的ATP提取效率更高,可显著提高仪器检测土壤样品的灵敏度和可靠性,因此可有效应用于各类土壤的微生物量的监测,为土壤环境监控提供微生物量的可靠数据。     

生物发光法微生物快速检测试剂的性质及其影响因素研究*

研究了ATP生物发光微生物快速检测试剂的反应动力学、反应最适温度、pH以及各种影响因素。在ATP生物发光反应中,测试系统中D-荧光素用量为40～50μg／mL时对反应已经足够;发光脉冲计数CPM值随反应时间的延长不断降低,开始的1min内,其CPM值下降最快,然后下降速度不断减缓;反应的最适温度为24℃-25℃;而体系的最佳pH为7．2—7．4。配制好的发光试剂溶液置于4℃保存45h,可以保持86％的活力,在25℃时保温1h,活力下降较少,随时间的增加,活力逐渐下降,到6．5h时,仅剩53．5％的活力,而     

东方蜜蜂微孢子虫腺苷酸激酶的生物信息学与基因表达特征

本研究旨在解析东方蜜蜂微孢子虫Nosema ceranae的腺苷酸激酶（Adenylat kinase, ADK）NcADK的理化性质和分子特性,并检测NcADK基因在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程中的表达特征,以期丰富NcADK相关信息,并为进一步的功能研究提供依据。利用相关生物信息学软件预测和分析NcADK的理化性质、信号肽、磷酸化位点、二级结构和三级结构。使用MEME软件和Batch CD-Search工具分别预测东方蜜蜂微孢子虫和其它7种微孢子ADK蛋白的保守基序和保守结构域。通过Mega 11.0软件基于ADK氨基酸序列构建进化树。采用RT-qPCR检测东方蜜蜂微孢子虫侵染过程中NcADK的相对表达量。结果表明,NcADK的CDS含有540个核苷酸,可编码179个氨基酸;NcADK的分子量约为20.66 kDa,分子式为C903H1488N258O278S8,脂肪系数为100.61,平均亲水系数为-0.502,等电点为6.83,含29个负电荷氨基酸和29个正电荷氨基酸;NcADK可同时定位于细胞质、线粒体、细胞核、囊泡和过氧化物酶体;NcADK含20个磷酸化位点,不含典型的信号肽;NcADK含88个α-螺旋,24条延长链,17个β-转角,50个无规则卷曲,与模板A0A0F9WEU7.1.A之间的序列同源性为100%;在东方蜜蜂微孢子虫、东方赤孢子虫Hamiltosporidium tvaerminnensis、肠脑炎微孢子虫Encephalitozoon intestinalis、按蚊微孢子虫Anncaliia algerae和角膜条孢虫Vittaforma corneae ADK中均鉴定到1个相同的结构域和5个相同的保守基序;东方蜜蜂微孢子虫与蜜蜂微孢子虫Nosema apis的ADK在进化树上聚为一支。相较于接种后1 d（1 day post inoculation, 1 dpi）,NcADK的表达量在2 dpi上调但无显著差异(P>0.05）,在3 dpi 和4 dpi均显著上调（P>0.05）。研究结果明确了NcADK的理化性质和分子特性,并揭示NcADK是潜在的亲水性蛋白和胞内蛋白,不同微孢子虫的ADK具有较强的保守性,东方蜜蜂微孢子虫及其姐妹种蜜蜂微孢子虫的ADK具有高同源性,NcADK在3 dpi和4 dpi被激活表达。     

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap₅A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl⁻ channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.     

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.     

The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains I and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the "closed" state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an "open" state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.     

生物发光法测定溶脲脲原体内微量ATP

目的　监测在液体培养基中生长的溶脲脲原体(Uu)细胞内微量ATP的变化趋势,方法　运用生物化学发光技术,建立ATP定量法。结果　Uu标准血清型1(U     

腺苷酸激酶与细胞凋亡

腺苷酸激酶(AK)在维持细胞能量平衡中起着重要作用,新近又发现它与细胞凋亡有着密切的关系.在凋亡过程中,AK2从线粒体膜间释放到胞浆是一种非常普遍的现象,但其在细胞凋亡中作用仍很不清楚.综述了近年对腺苷酸激酶的研究进展,探讨了腺苷酸激酶在细胞凋亡程序中所扮演的角色.     

Intracellular ATP Levels Affect Secondary Metabolite Production in Streptomyces spp.

The addition of extracellular ATP (exATP) to four Streptomyces strains had similar effects: low exATP levels stimulated antibiotic production and high levels reduced it. Compared with antibiotic production, the concentrations of intracellular ATP (inATP) in the tested strains were opposite, which suggests a role of inATP in regulating secondary metabolite production. Under inactivation of the polyphosphate kinase gene (ppk) in Streptomyces lividans, we observed the same results: when the inATP level in the mutant strain was lower than in the parent strain, more antibiotic was produced. Combining all the results, a strong inverse relationship between [inATP] and the secondary metabolite production is suggested by this study.     

Kinetin effect on ATP synthesis and on adenylate kinase activity in pea seeds

Pea seed powder incubated in the presence of AMP and phosphoenolpyruvate (PEP) accumulated relatively large amounts of ATP. The rate of accumulation in