NASBA荧光分子信标技术定量检测丙型肝炎病毒

建立NASBA荧光分子信标探针检测技术,并对国家HCV标准品、人工构建HCVRNA野生株及HCV抗体阳性不同人群进行检测。实验结果:该方法检测HCV的灵敏度为103拷贝ml血清,阴性参比品的符合率为100%;检测的线性范围为103拷贝～109拷贝ml血清;精密性(CV值)小于6%,在HCV抗体阳性人群中HCVRNA的检出率在45%～65%之间。结论:该方法在HCVRNA临床定量检测中具有良好的灵敏度、特异性、重复性与实用性。     

Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis

Molecular Biology Reports - RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key...     

应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

HCVRNA反转录PCR技术的建立及对血液样品的检测

实验建立了ＨＣＶ　ＲＮＡ的反转录和套式ＰＣＲ技术,扩增出２３２ｂｐ的核酸片段,经酶切电泳图谱和Ｓｏｕｔｈｅｒｎ杂交鉴定,来自ＨＣＶ基因５,端非编码区。实验从抗ＨＣＶ阳性的１８例血浆样品和１９例血清样品中分别检出７例和１３例ＨＣＶＲＮＡ阳性。     

Determination of polyadenylate-rich ribonucleic acid in the nucleus and in the cytoplasm of plasmacytoma cells.

A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values. The binding of poly(A)-containing RNA to the filter exhibited a broad pH-dependence compared with that of rRNA. The ratio of poly(A)-rich RNA/rRNA retained by the filter was maximal between pH7 and 8. The presence of 1 mM-EDTA or a high concentration of NaCl (over 0.5M) decreased the affinity of RNA for the filter. The amount of poly(A)-containing RNA in the nucleus and in the cytoplasm of a plasmacytoma cell line (MPC-11) labelled with [32P]Pi was determined by the Millipore-filter technique under conditions that minimized contamination by rRNA. These data were compared with the estimations made by oligo(dT)-cellulose chromatography. The results obtained by these two methods were in good agreement for RNA labelled for short periods (up to 2h). In long labelling and pulse-chase experiments, however, contamination of the filter by rRNA of increasing specific radioactivity in the cytoplasm gave an erroneous value for poly(A)-containing RNA by the Millipore-filter technique. Determinations made on the nuclear fraction by these two methods did not show significant variation in short- and long-term labelling experiments.     

Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.     

A型流感病毒的反向遗传技术研究进展

A 型流感病毒基因组为单股负义RNA,分为8 个片段。反向遗传技术即从克隆的cDNA 产 生病毒的过程,是研究RNA 病毒、也是研究A 型流感病毒基因结构与功能的新技术。介绍了A 型流感病毒反向遗传技术的发展,完全以质粒为基础的新操作系统及其在研究病毒的生命周期、 致病性、产生基因修饰的疫苗候选株等方面的应用。     

A micromethod for measuring the molar concentration of polyadenylated RNA in the presence of ribosomal RNA

A method has been developed for measuring the molar concentration of RNA and the mole fraction of polyadenylated RNA. Using known mixtures of globin mRNA and rRNA composed of 20 to 85% rRNA, the molar concentration of globin mRNA, a polyadenylated species, was determined in 45 min, with the consumption of less than 100 ng of total RNA. The technique is particularly well suited for determining the molar concentration of poly(A)+ RNA after chromatographic enrichment in columns of oligo(dT)-cellulose or poly(U)-Sepharose. The method makes possible the adoption of a molar standard.     

Improved methods for the delivery of liposome-sequestered RNA into eucaryotic cells

RNA sequestered by negatively charged liposomes becomes cell-associated following interaction between eucaryotic cells and the liposomes. This paper provides evidence that cell-associated RNA is internalized by the cells. In fact, (a) when Escherichia coli and mammalian RNA are entrapped within the same liposome population and delivered into cultured cells, one can observe degradation of the procaryotic but not the eucaryotic RNA. Such an event cannot happen extracellularly. (b) Scanning electron microscopy reveals no more than 10 liposomes adhering to each cell upon liposome-cell interaction under conditions in which the RNA entrapped by 140 liposomes becomes associated with each cell. The ability of liposomes prepared by (a) the cochleate process, (b) the reverse-phase evaporation technique, and (c) the ether infusion technique, to sequester and deliver RNA into cells was investigated. Reverse-phase evaporated liposomes were most efficient in sequestering RNA (20–40%), however, all types of liposomes delivered RNA with comparable efficiency. The rate of liposome-mediated RNA delivery into mammalian cells could be substantially improved when: (a) liposome-cell interaction was carried out at pH 6.5 (twofold increase over pH 7.5), (b) a basic protein (methylated albumin) was present (two- to threefold increase), (c) liposome-cell cultures were treated with polyethylene glycol 6000 (four- to eight-fold increase), and (d) DEAE-dextran was added during interaction of liposomes with cell monolayers (four- to eight-fold increase).     

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique.

A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.     

不同检测策略在预测非小细胞肺癌的PD1/PD-L1抑制剂临床疗效中的应用

近10年来,程序性死亡因子1(programmed death-1,PD-1)及其配体(programmed death ligand-1,PD-L1)的抑制剂在非小细胞肺癌(non-small cell lung cancer,NSCLC)的临床治疗中取得了重大突破,有望改变晚期NSCLC的治疗方式。然而,PD-1/PD-L1抑制剂在对NSCLC的治疗中需要借助有效的生物标志物以寻找受益人群(约20%～40%)。目前,临床上主要的判断标准是PD-L1的表达水平。本文综述了近年来在NSCLC中,与预测PD-1/PD-L1抑制剂疗效的PD-L1表达相关的检测方法,包括免疫组化、基于DNA/RNA水平检测、可溶性PD-L1的检测、正电子发射断层显像(positron emission tomography,PET)技术、多重免疫组化技术、流式细胞术和液体活检技术等,着重探讨了不同检测策略在评价PD-L1表达上的最新进展及应用前景,从而推动其在NSCLC免疫治疗中的临床应用。     

Red cell ghost-mediated microinjection of RNA into HeLa cells: I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA

We have compared two techniques for introducing RNA into red blood cell ghosts. In the pre-swell technique, RNA is introduced into red cells without prior removal of endogenous contents. In the multiple lysis technique, the red cells are subjected to two or three cycles of reversible lysis, prior to introducing the RNA, in order to first remove the normal red cell constituents. The pre-swell technique offers much greater entrapment of both tRNA and protamine messenger RNA (mRNA), but the RNA appears to be degraded during the procedure. This may be due either to nuoleolytic degradation or oxidation by the high concentration of endogenous hemoglobin. The multiple lysis technique offers much lower entrapment but also results in diminished degradation of the entrapped RNA. Although some degradation is apparent, a significant portion of the biological activity of the entrapped protamine mRNA is retained. We have also fused red cells loaded with protamine mRNA by the multiple lysis technique to HeLa cells using polyethylene glycol 6000. The recipient HeLa cells are capable of translating this heterologous message into protamine, a trout testis chromosomal protein.     

Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Replication of Bacteriophage Ribonucleic Acid: Analysis of the Ultrastructure of the Replicative Form and the Replicative Intermediate of Bacteriophage R17

A detailed qualitative and quantitative comparison was made of the ultrastructure of single-stranded ribonucleic acid (RNA) from bacteriophage R17 and double-stranded replicative form (RF) and replicative intermediate (RI) from cells infected with this bacteriophage. The nucleic acids were prepared for electron microscopy by the protein monolayer spreading technique of Kleinschmidt. Single-stranded RNA aggregated during spreading in the absence of urea, whereas RF and RI did not. On the other hand, RF and RI appeared to be susceptible to shear during spreading, whereas R17 RNA was not. From the maximal length of RF, a base translation of 3.14 A was calculated. This value favors a 10-fold helix model of double-stranded RNA. The same base translation was found for R17 RNA, indicating a stacked base structure for single-stranded RNA spread in the presence of urea. RI is a branched structure and the branches are removed by ribonuclease treatment. The branches are believed to be nascent single-stranded viral RNA. The contour length of the branch was equal to the contour length of the main chain up to the branch point, as predicted from theoretical analysis of the replication of viral RNA. The structure of RF and the main chain of RI was also analyzed by plotting the log (end-to-end distance squared) versus log (contour length). This demonstrated structures intermediate in stiffness between a random coil and a rigid rod.