Direct detection of unamplified hepatoma upregulated protein RNA in urine using gold nanoparticles for bladder cancer diagnosis

ObjectiveTo develop a gold nanoparticle (AuNP) assay for direct detection of unamplified HURP RNA in urine.Design and methodsHURP RNA was extracted from urine samples (50 bladder carcinoma patients, 25 benign bladder lesions, and 25 controls) and further purified using magnetic nanoparticles (MNPs), functionalized with HURP RNA-specific oligonucleotides, and then detected by RT-PCR or gold nanoparticles.ResultsThe developed HURP RNA AuNP assay has a sensitivity and a specificity of 88.5% and 94%, respectively, and a detection limit of 2.4 nmol/L. The concordance between the HURP AuNP assay with RT-PCR after RNA purification using functionalized MNPs was 97%.ConclusionsThe developed colorimetric HURP RNA AuNP assay is sensitive, simple, and can aid noninvasive diagnosis of bladder cancer.     

两种巢式PCR检测慢性粒细胞白血病患者骨髓移植后微小残留病的比较

目的　比较 2种巢式聚合酶链反应 (PCR)检测骨髓移植后慢性粒细胞白血病 (CML)患者微小残留病。方法　分别用一步法逆转录 PCR(RT PCR)和两步法RT PCR 2种巢式PCR检测骨髓移植后CML患者BCR ABL融合基因 ,并做 2种方法的灵敏度实验。结果　 2种巢式PCR灵敏度实验中 ,一步法RT PCR和两步法RT PCR的巢式PCR可分别检测出 0 .1和 1pgK5 6 2细胞总RNA中的BCR ABL融合基因 ;同时对 19份来自 12例骨髓移植后CML患者标本检测 ,一步法RT PCR的巢式PCR检测出阳性的最长移植时间为 6 90d ;两步法RT PCR的巢式PCR为 4 15d。结论　一步法RT PCR的巢式PCR检测骨髓移植后CML微小残留病具有较高灵敏度 ,操作简便快速 ,特别适用于临床检测。     

Application of the PCR technique for a rapid,specific and sensitive detection of Salmonella spp. in foods

We report here the use of a PCR based assay modified by us for the detection of Salmonella spp. in foods, based on amplification of a 236 bp Salmonella specific hin/H2 region [Way et al. (1993) Applied and Environmental Microbiology 59, 1473-1479], using Ampli Taq Gold polymerase. Using this assay we were able to detect all the Salmonella serovars tested. The limit of detection was 1 fg of purified target DNA or N x 10(0) (1-3 cells) cfu ml(-1) of pure bacterial culture. This assay could detect N x 10(0) cfu Salmonella cells g(-1) of the food sample unambiguously in presence of endogenous microflora following 6 h enrichment, thus requires a duration of approximately 10 h for the full processing from DNA template preparation, PCR and visualization of DNA product on agarose gel. The main advantage of this PCR detection method is its sensitivity, and specificity. We also tried to adopt DNA template isolation method simply by boiling the bacterial cells thereby reducing the possibility of contamination, cutting the processing time and cost considerably. This can be an added advantage for the use of this system in simple lab setups.     

A new combination of RNA-mediated DNA ligation and on-chip elongation for detecting viral RNA

We describe a novel method that combines RNA-mediated DNA ligation and on-chip elongation for detecting viral RNA. These virus species-specific detection probes (DPs) were designed to match sequences of the "target" virus and then chemically synthesized into 2 parts. If the target virus exists, 2 parts of the DP can be ligated by T4 DNA ligase. The ligated DP was hybridized to its corresponding capture probe (CP) on a DNA array. Then, an on-chip DNA polymerization (including Cy3-dUTP) was performed using the ligated DP as a template and the CP as a primer, which resulted in a reporting fluorescent signal. If the target virus does not exist in a sample, no fluorescence signal is produced. Four common tobacco viruses, tobacco mosaic virus, cucumber mosaic virus, potato virus Y, and potato virus X in single and mixed infections were successfully detected by this method. The sensitivity of the detection limit of this assay is 10 times higher than that of ELISA. The minimum dilution detection limit of this assay was 10(-4) (infected sap/healthy sap). The method has the potential to detect any viral RNA from animal, germs, or fungi where the sequence is known.     

肠道病毒71型RT-LAMP快速检测方法的建立及初步应用

目的建立手足口病肠道病毒71型(EV71型)逆转录环介导等温扩增(RT-LAMP)快速检测方法,并对其进行应用评价。方法利用软件针对EV71型病毒特异性基因6个区域设计4条LAMP引物,在普通恒温水箱内65℃保温约1 200min完成RT-LAMP扩增,扩增结果通过电泳和肉眼来判断。利用RT-LAMP和逆转录聚合酶链反应(RT-PCR)方法同时检测70份EV71型肠道阳性标本;将EV71型的RNA做一系列10倍稀释后用RT-LAMP和RT-PCR方法进行检测来比较两者敏感度。结果 EV71型出现LAMP特征性梯状条带,肉眼可以判断结果;RT-LAMP与RT-PCR方法检测100份EV71型标本的检出率比较差异无统计学意义(P0.05);RT-LAMP方法的敏感度(10.0pg/μL)与RT-PCR方法相同。结论建立了一个可以在普通恒温水箱内进行核酸扩增的EV71型RT-LAMP检测方法,初步应用证实该方法有一定的应用前景。     

Development of an EvaGreen based real-time RT-PCR assay for rapid detection,quantitation and diagnosis of goose calicivirus

An unclassified calicivirus (CV) detected in geese was recently reported and proposed as a new member of the family Caliciviridae. There is limited information about the epidemiology, etiology and detection method of goose-origin CV (GCV) to date. In this study, an EvaGreen based fluorescence quantitative real-time RT-PCR assay was developed and optimized for the detection of GCVs. The assay sensitively detected GCV RNA template with a good linear standard curve. We also demonstrated the specificity and reproducibility of the detection method for GCVs. Thus, the method developed in this study will benefit the investigation of possible sporadic outbreaks of CV infections in geese, as well as epidemiological and etiological studies of GCVs.