一种用于核酸高灵敏检测的液滴式数字聚合酶链式反应芯片

为了实现对核酸的高灵敏度检测,构建了一种新型的液滴式数字聚合酶链式反应(dd PCR)芯片.该芯片由产生液滴的聚二甲基硅氧烷(PDMS)模块和储存液滴的玻璃腔室构成.实验结果表明,该芯片可以在25 min内产生2×106个直径为20μm的微液滴(体积4.187 p L).利用该芯片定量检测了表皮生长因子受体(EGFR)基因第19号外显子,在DNA浓度为106~101copies/μL范围内呈现良好的线性关系(R2=0.9998);在浓度为106copies/μL的19号外显子野生型DNA中检测105~100copies/μL的突变型DNA,其检测敏感度可达到0.0001%.该方法在同一芯片上实现了液滴产生、核酸扩增和荧光信号读取的功能,在核酸绝对定量及痕量突变基因的检测中具有潜在应用前景.     

Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence

Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter 12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated before Au-NPs are included in any qPCR assay. Figure Raw amplification profiles in the presence and absence of gold nanoparticles     

流动型微流控PCR扩增芯片的研究

组装了由注射泵进样系统、微流控芯片和三温区加热器组成的流动型PCR扩增系统,该系统具有扩增速度快、交叉污染小、芯片可重复使用和操作方便等特点.优化了芯片厚度、隔热材料和流速等影响PCR扩增的因素.在4.9min内经24个循环成功地扩增了浓度为1ng/100μL的λ-DNA(500bp).     

The effect of heat treatment on the detection of peanut allergens as determined by ELISA and real-time PCR

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitised individuals. The detection of peanut traces in food products is therefore of prime importance. Peanut traces which can be (unintentionally) present in food products have usually undergone one or more processing steps like roasting and baking. Therefore, methods designed to detect such traces have to be capable of detecting heat-treated peanuts. Commonly used methodologies designed to detect peanut traces in food products are enzyme-linked immunosorbent assays (ELISAs) that detect peanut-specific proteins, and polymerase-chain-reaction (PCR)-based methods targeting peanut-specific DNA. A comparative analysis of such methods was performed and the impact of heat treatment on peanut kernels as well as the impact on a peanut-containing food matrix are investigated. Our results show that heat treatments have a detrimental effect on the detection of peanut with either type of method and that both types of methods are affected in a similar manner.     

Studies on the response of soil microflora to the application of the fungicide fenhexamid

The aim of this work was to evaluate the impact of the fungicide fenhexamid (FEX) on the genetic structure of soil bacterial communities using the Ribosomal Intergenic Spacer Analysis molecular technique. Using real-time PCR, we also tried to quantify the pcaH sequences which encode the dioxygenases involved in the degradation process of a variety of aromatic compounds. Soil taken from a vineyard in the Etna Park (Sicily, Italy) was treated with FEX in the ratio 2?µg?g^?1 soil every 7?days, the process being repeated four times. The analyses were carried out before treatment and 7?days after each further application of FEX. At the same time, the degradation rate was evaluated. The use of FEX determined a variation in the bacterial component of the soil which could be seen in an increase of some microbial strains and the inhibition of others. The pcaH sequence was already present in the genes of the soil microrganisms studied, but the use of FEX increased the number of the gene copies. These results suggest that the microbial population of the soil adapted to the presence of FEX with an increase in degradation potential. The measurements of the extent to which FEX was degraded confirm this hypothesis, showing that the molecule disappeared more quickly with successive applications.     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection     

应用于氧还原电极的新型Co-PAn-C复合催化材料

借鉴美国Los Alamos国家实验室报告的应用于燃料电池氧电极的新型Co-PPy-C (PPy, polypyrrole)复合物催化材料, 提出Co-PAn-C (PAn, polyaniline)复合材料可能对氧的电化学还原也具有催化活性, 并首次制备出Co-PAn-C复合催化材料. 发现Co-PAn-C对氧还原有显著的催化效果, 碱性介质中氧气气氛下, 电极电位为－0.2 V vs. Hg/HgO时电流密度达到128 mA•cm^－2, 性能也比较稳定. 初步研究了Co-PAn-C对ORR (oxygen reduction reaction)的催化机理, 可能是在结构中形成了Co-N活性位置, 影响了氧和反应中间产物在电极上的吸附和脱附过程.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

An Activatable NIR Fluorescent Probe for NAD(P)H and Its Application to the Real-Time Monitoring of p53 Abnormalities In Vivo

NAD(P)H is crucial for biosynthetic reactions and antioxidant functions. However, the current probes developed for detecting NAD(P)H in vivo require intratumoral injection, which limited their application for animal imaging. To address this issue, we have developed a liposoluble cationic probe, KC8 , which exhibits excellent tumor-targeting ability and near-infrared (NIR) fluorescence after reaction with NAD(P)H. By using KC8 , it was demonstrated for the first time that the level of NAD(P)H in the mitochondria of living colorectal cancer (CRC) cells was highly related to the abnormality of the p53. Furthermore, KC8 was successfully used to differentiate not only between tumor and normal tissue but also between tumors with p53 abnormality and normal tumors when administered intravenously. Finally, we evaluated tumor heterogeneity through two fluorescent channels after treating a tumor with 5-Fu. This study provides a new tool for real-time monitoring of the p53 abnormality of CRC cells.