数字PCR技术在核酸标准物质研制中的应用

在标准物质研制领域,生物标准物质的研制逐渐成为了研究热点,同时基于核酸的检测技术的开发与应用又推动了核酸标准物质的进程。核酸标准物质需要高级别、精准的定值方法,数字PCR作为单分子定量技术得到了广泛的应用。数字PCR是一种测定核酸分子的绝对定量方法,如微滴式数字PCR是采用油包水形成的微滴作为反应室,将含有DNA模板的反应溶液分配到大量独立的反应室中进行扩增反应,再通过统计反应室中的阳性信号来定量DNA的拷贝数,从而达到精确定量核酸拷贝数的目的。综述了近年来关于数字PCR及其在核酸标准物质研究领域的最新应用进展,重点综述了其在转基因检测、医疗诊断等领域的应用进展,以期为核酸标准物质的研制提供参考。     

核糖核酸标准物质研制与应用进展

核糖核酸(RNA)标准物质是保证核酸体外诊断数据准确一致的重要实物标准,广泛用于病毒类疾病的检测、仪器校准、试剂盒评价及能力验证等工作中,其目的是保证测量过程和测量结果的准确一致。自1997年,WHO研制出了首个针对核酸扩增技术的丙型肝炎核酸国际标准物质,使全球不同实验室、不同方法检测的结果有了可比性。截至2023年,中国在国家标准物质资源共享平台发布的RNA有证标准物质有近百种,对于RNA标准物质的研究与应用,日益受到世界各国计量机构、相关国际学术组织与企业的重视,尤其是新型冠状病毒疫情暴发后,RNA标准物质成为了生物标准物质的研制热点。随着标准物质研制技术的不断发展,标准物质的量值准确性也有所提升,为测量结果的准确一致提供保障。通过对当前RNA标准物质的制备方法、分装、定值技术、不确定度评定及应用领域进行综述,为RNA标准物质的研制工作以及未来在临床医学、农业生产、动物医学、食品安全等领域的应用提供参考。     

质量平衡法及其在标准物质定值中的应用进展

为保证不同地区、不同时间测量结果的可比性,测量结果需溯源至适当的、规定的参考标准。对于化学、生物、工程、物理学领域的材料和样品测量,该参考标准为标准物质。由此可见,标准物质的定值对物质的检测及定量是十分重要的。标准物质（reference material,RM）是一种足够均匀的、具有一种或多种相对容易确定的特性值的材料或物质,可用于给材料赋值、评价测量方法及校准测量仪器等。质量平衡法作为标准物质的定量方法之一,是一种常用的纯度测量方法,将水分、灰分、挥发组分、无机元素等杂质的含量从100%中扣除,再根据主要组分在有机组分中的百分比来确定物质纯度。质量平衡法具有较高准确度,能够溯源到国际单位制中的质量单位,且若使用基准方法测量样品中的主成分及各部分杂质以完成整个质量平衡法的测量,质量平衡法则有望成为新的基准方法。基于此,对质量平衡法原理及质量平衡法在标准物质的研制中的应用进行了介绍,并对近期质量平衡法在标准物质中的最新应用进行了总结,以期探索质量平衡法在标准物质研制中的更多可能。     

尿素(UREA)血清标准物质的制备及定值

采用新鲜人血为原材料,制备成具有UREA标准浓度(5.32±0.16)mmol/L的血清标准物质,采用分光光度法对此标准物质进行定值,并且对UREA浓度含量进行不确定度评定,均匀性稳定性检验。     

转基因定量检测用质粒分子标准物质研究进展

近10年来,应用于转基因植物产品定量检测的质粒分子标准物质凭借其易于富集,快捷高效等优点,成为各国检测机构的研究热点。对国内外转基因植物核酸定量检测用的质粒分子标准物质研究进展进行总结和评述,并且对该类标准物质在研制过程中的问题进行分析,同时展望了该类标准物质的发展与应用。     

T淋巴细胞亚群CD4+比例标准物质的研制

CD4⁺ T细胞的减少常见于恶性肿瘤、遗传性免疫缺陷症、艾滋病等。检测实验室常需要对CD4⁺ T细胞检测能力进行质控,急需有准确CD4⁺比例量值的T淋巴细胞标准物质。将人工制备的T淋巴细胞进行纯化后,使用流式细胞术进行标准物质候选物的均匀性检验、稳定性检验。联合8家检测实验室采用流式细胞微球计数法对标准物质候选物进行协同定值。实验数据统计表明,该标准物质候选物的均匀性好,并且在-20 ℃储存条件下可稳定12个月以上,其标准量值CD4⁺比例为74.0%±6.7%（k=2）。该标准物质适用于流式细胞仪中CD4⁺细胞占总淋巴细胞的百分比项目的计量校准,以及流式细胞术检测过程的方法验证和检测结果的质量控制。     

人D6S1043-1型STR质粒DNA标准物质的研制

短串联重复序列（short tandem repeat,STR）是存在于人类基因组中的一类具有长度多态性的DNA序列,由含2~6个碱基对的重复单位串联构成。DNA STR分型检验是当前法庭科学进行个体识别和亲缘鉴定的主要依据。STR分型标准物质是DNA STR检验量值溯源的基础和关键物质,对其进行研究将极大推动我国法庭科学DNA STR检验的标准化进程。以STR基因座D6S1043为例,介绍人类基因组中STR序列的质粒DNA标准物质的制备过程。首先,合成包含13个重复单元（[ATCT]13）的D6S1043序列（定义为D6S1043-1）并将其与pUC57载体连接构建重组质粒,制备质粒溶液。其次,通过酶切鉴定、Sanger测序、多种STR分型试剂盒的分型鉴定等方法检验DNA序列的准确性。再次,采用微滴式数字PCR方法（droplet digital PCR,ddPCR）检测质粒浓度,评估质粒溶液的均匀性和稳定性。最后,由8家实验室协作,测定浓度标准值,综合分析均匀性检验、稳定性检验、定值过程等引入的不确定度,得到总不确定度;由6家实验室协作,测定D6S1043-1的分型值。结果表明：该标准物质的均匀性、稳定性良好,在-20 ℃可保存6个月,在4 ℃可保存14 d;核酸拷贝数为（7.7±1.2）×103 copies·μL-1;在D6S1043基因座上的分型值为13。人D6S1043-1型STR质粒DNA标准物质 [编号：GBW（E）091072] 是我国法庭科学DNA检验领域首批有证标准物质之一,其研制过程为其他STR基因座的质粒DNA标准物质的研制提供了一定的借鉴。     

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7×10^–3 cm s^–1 at 10°C, 1.2×10^–3 cm s^–1 at 15°C, 1.4×10^–3 cm s^–1 at 20°C, 1.8×10^–3 cm s^–1 at 25°C, 2.1×10^–3 cm s^–1 at 30°C and 3.5×10^–3 cm s^–1 at 37°C. The mean value of the activation energy of water diffusion (E_a,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS-inhibited erythrocytes. The values of P and E_a,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.     

顺磁物质对土壤低场核磁共振信号特征及含水量测定的影响

核磁共振(NMR)技术由于具有高效快速、不破坏土壤结构且对人体无害等优点,逐渐被应用到土壤学相关领域研究中。然而,土壤中顺磁物质的存在对核磁共振信号特征的影响仍不明确。本研究旨在揭示顺磁物质对不同类型土壤低场核磁共振(LF-NMR)信号特征和土壤含水量测定的影响。结果表明:土壤水的LF-NMR信号量最高可达150左右,土壤矿物、有机质和微生物等固相物质的LF-NMR信号量基本不超过0.3,相对可以忽略。质地和顺磁物质对土壤水的LF-NMR信号量测量有更大影响。LF-NMR仪器存在弛豫时间监测盲区,信号量损失主要是由于顺磁物质加速了水中氢质子的弛豫过程,导致小孔隙中水分反馈的极快的LF-NMR信号不能被监测设备捕获。对于顺磁物质含量较少的壤性潮土(1.2%)和黏壤性黑土(1.3%),LF-NMR信号量损失不大,其与土壤含水量呈线性关系;但对于黏粒含量(45.3%)和顺磁物质含量(4.0%)较高的黏性红壤,测定中会损失一部分LF-NMR信号量,监测到的LF-NMR信号量与土壤含水量不再呈线性关系。此外,外源添加顺磁物质(3.0 g·L^-1的MnCl₂溶液)也会降低黑土和红壤中可被监测的LF-NMR信号量,黑土和红壤的信号量最大损失率分别为41.0%和46.7%,极大地改变其与土壤含水量之间的定量关系。因此,在利用LF-NMR测量富含顺磁物质(>1.3%)或有外源顺磁物质进入的黏性土壤的含水量时,应先通过校正降低顺磁物质等对LF-NMR信号量的影响。研究结果对利用低场核磁共振技术准确分析土壤水分分布及土壤孔隙结构具有重要意义。     

Pyrosequencing-based analysis of bacterial community and metabolites profiles in Korean traditional seafood fermentation: a flatfish-fermented seafood

Bacterial community and metabolites were analyzed in a flatfish jeotgal, a Korean fermented seafood. Inverse relationship of pH and 16S rRNA gene copy number was identified during fermentation. Lactobacillus was the predominant bacterial genus. Increase of Firmicutes was a common characteristic shared by other fermented seafood. Fructose, glucose, and maltose were the major metabolites.     

Observation of carbon labelling in cell metabolites using proton spin echo NMR

A heteronuclear spin echo experiment is described which allows detection of both ¹²C and ¹³C labelled species in a ¹H spectrum. Fractional labelling of ¹³C labelled metabolites can thus be observed. The method is illustrated with a study of the exchange of ¹³C label between the methyl groups of alanine and pyruvate catalysed by the enzyme alanine aminotransferase (E.C. 2.6.1.2) both in the human erythrocyte and $\text{in}\text{,}\text{vitro}$.     

A study of the interactions among adenosine triphosphate, epinephrine, and magnesium ions

The interactions among adenosine triphosphate, Mg⁺², and epinephrine at pH's below 7.0 have been studied by observing the effects of these interactions on the chemical shifts and line widths of their ¹H and ³¹P nuclear magnetic resonance spectra. Mg⁺² is tightly bound by the β- and γ-phosphate groups of adenosine triphosphate and there is a weak association between this chelate and epinephrine. In the ternary complex, the aromatic ring of epinephrine overlaps the purine ring of adenosine triphosphate and there appears to be an ionic interaction between the protonated amino group and the α-phosphate of adenosine triphosphate. It was also found that dichloroisoproterenol forms essentially the same type of ternary complex.     

Noninvasive skeletal muscle lactate detection between periods of intense exercise in humans

We investigated whether localized ¹H nuclear magnetic resonance spectroscopy (NMRS) using stimulated echoes (STEAM) with a long mixing time (t _m) allowed the suppression of the fat signal and detection of lactate in skeletal muscle. The ¹H NMRS sequence was first validated in three isolated and perfused rabbit biceps brachii muscles. Spectra were obtained on a wide-bore spectrometer using a dual-tuned probe (¹H and ³¹P). Death was simulated by ceasing the muscle perfusion, which allowed post-mortem changes to be followed. During and after the simulated death, changes in levels of pH and in content of energy-rich compounds were observed with ³¹P NMRS. Our results showed an inverse linear relationship between pH and lactate in each of the three rabbits (r = 0.93, P < 0.001; r = 0.92, P < 0.01; r = 0.89, P < 0.01) and a decrease in phosphocreatine and concomitant increase in lactate. We then investigated whether this sequence allowed repeated detection of lactate in human soleus muscle during the recovery between periods of intense exercise (force-velocity test, F-v test). Seven subjects mean age 25.1 (SEM 0.8) years participated in this study. Soleus muscle lactate was detected at rest and for 3 min 30 s of the 5-min recovery between periods using a 2.35-T 40-cm bore magnet spectrometer. Arm venous plasma lactate concentration was measured at rest, during the F-v test when the subject stopped pedalling (S₁), and at the end of each 5-min recovery between periods (S₂). Results showed that the venous plasma lactate concentration at S₁ and S₂ increased significantly from the beginning of the F-v test to peak anaerobic power (W _an,peak) (P < 0.001). The spectra showed that muscle lactate resonance intensity rose markedly when W _an,peak was achieved. The muscle lactate resonance intensity plotted as a percentage of the resting value increased significantly at W _an,peak compared with submaximal braking forces (P < 0.05). We concluded from these results that localized ¹H NMRS using STEAM with a long t _m allows suppression of the fat signal and repeated detection of lactate on isolated perfused skeletal muscle in animals and between periods of intense exercise in humans. Accepted: 19 January 1998     

Seasonal variation in the composition of aquatic organic matter in some Nova Scotian brownwaters: a nuclear magnetic resonance approach

Using a novel concentration technique (reverse osmosis and freeze-drying) as well as a standard analytical technique little used with limnological samples (solid state ¹³C nuclear magnetic resonance), we studied the chemical structure of aquatic organic matter from four closely located freshwater sites in Nova Scotia. The main conclusions drawn from the data are that: (a) the aromatic C fraction which is assumed to be refractory remains at less than 10% of the total, with a slight increase in relative importance in the fall, (b) less structurally complex aliphatic carbon decreases from winter to spring and remains at lower levels into later fall, (c) carbohydrates are at a maximum during the summer, (d) the carboxylic C fraction is at a minimum in the summer and maximum in the fall and winter. Results show roughly the same annual patterns of C composition for the two running water sites. Our data suggest that the hydrological processes which carry terrestrial and wetland DOM to streams and lakes allow a selective adsorption process of DOM to occur in soils. We compare our data to those from other freshwater studies and suggest that the importance of aromatic carbon in freshwaters has probably been overestimated in the past, with a corresponding underestimate of the more biologically labile carbohydrate and aliphatic material.Department of Chemistry, McMaster Universtiy