Europium(III) modified silicone nanoparticles for ultrasensitive visual determination of tetracyclines by employing a fluorescence color switch.

Silicon nanoparticles (SiNPs) modified with Eu(III) were synthesized and are shown to be a viable ratiometric fluorescent probe for tetracycline antibiotics. SiNPs/Eu under 405 nm excitation display two emissions, viz. a strong cyan colored fluorescence peaking at 497 nm and a weak pink fluorescence peaking at 622 nm. On addition of tetracyclines (chlortetracycline, tetracycline, doxycycline), the fluorescence at 497 nm is reduced, while the one at 622 nm is increased. Thus, the visible color of fluorescence changes from cyan to pink. This was exploited to design ratiometric fluorometric method for detecting tetracyclines. The method has a limit of detection that is lower by a factor of about 1000 when compared to the use of SiNPs only. A test paper was prepared with the SiNPs/Eu and then applied for the visual semi-quantitative detection of tetracyclines. With the addition of tetracyclines, the test paper exhibited a dosage-sensitive color conversion from cyan to pink with a visually discernible scale as low as 0.4 µM. Graphical abstract Tetracyclines decrease the fluorescence at 497 nm of europium (III) modified silicon nanoparticles (SiNPs/Eu) due to the inner filter effect and increase the one at 622 nm due to an antenna effect. Thus the fluorescence color of SiNPs/Eu changes from cyan to pink. Based on this color switch, a ultrasensitive and visual determination strategy for tetracyclines is proposed.

Application of high-resolution melting analysis for authenticity testing of valuable Dendrobium commercial products.

BACKGROUND: The accurate identification of botanical origin in commercial products is important to ensure food authenticity and safety for consumers. The Dendrobium species have long been commercialised as functional food supplements and herbal medicines in Asia. Three valuable Dendrobium species, namely Dendrobium officinale, D. huoshanense and D. moniliforme, are often mutually adulterated in trade products in pursuit of higher profit. RESULTS: In this paper, a rapid and reliable semi-quantitative method for identifying the botanical origin of Dendrobium products in terminal markets was developed using high-resolution melting (HRM) analysis with specific primer pairs to target the trnL-F region. The HRM analysis method detected amounts of D. moniliforme adulterants as low as 1% in D. huoshanense or D. officinale products. CONCLUSION: The results have demonstrated that HRM analysis is a fast and effective tool for the differentiation of these Dendrobium species both for their authenticity as well as for the semi-quantitative determination of the purity of their processed products. © 2017 Society of Chemical Industry.

The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System.

Background: Urinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours. Methods: We designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing. Results: UTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method. Conclusions: UTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.

A New Multiplex Genetic Detection Assay Method for the Rapid Semi-Quantitative Detection of Six Common Curable Sexually Transmitted Pathogens From the Genital Tract.

Background: Sexually transmitted infections (STIs) are some of the most common communicable conditions and exert impact on the health and lives of many hundreds of millions of people across the world every year. Screening high-risk populations and conducting comprehensive detection tests would lead to a significant improvement in preventing the transmission of STIs and help us to provide rapid treatment to those affected. Here, we successfully established and validated a novel high-throughput multiplex gene detection system (HMGS) for the simultaneous and semiquantitative detection of six important curable sexually transmitted pathogens in a single reaction from secretions samples. Method: Fluorescently labeled primers were designed to target specific conserved and single-copy gene fragments of Ureaplasma urealyticum (U. urealyticum), Mycoplasma hominis (M. hominis), Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N. gonorrhoeae), Trichomonas vaginalis (T. vaginalis), and Treponema pallidum (T. pallidum). The specificity and sensitivity of the STI-HMGS was validated and optimized using plasmids and quantitative genomic DNA. Next, we validated the performances of the STI-HMGS for clinical application by testing samples of clinical secretions collected from patients who visited the gynecology and urology outpatient clinics of our reproductive medicine center. Results derived from the STI-HMGS were then compared with three approved commercialized kits that used to detect U. urealyticum, C. trachomatis and N. gonorrhoeae, respectively, followed by further validation with Sanger sequencing for all pathogens. Finally, a comprehensive analysis of epidemiology was performed among different subgroups to investigate the association between infection rates and clinically-relevant information. Results: The sensitivity of STI-HMGS for six target genes was 10 copies/µL. Data derived from the detection of 381 clinical secretions demonstrated that the STI-HMGS exhibited high concordance rate compared with approved commercialized kits and almost 100% sensitivity and specificity for the detection of six sexually transmitted pathogens when validated by Sanger sequencing. Semi-quantitative analysis found that STIs caused by N. gonorrhoeae had a significantly higher (P<0.05) pathogen load than the other pathogens. Infections caused by C. trachomatis were significantly more common in younger individuals (P<0.05). We also found that U. urealyticum infections were more likely to happen in females; while the males were more affected by N. gonorrhoeae (P<0.05). Conclusions: STI-HMGS proved to be an efficient method for the semi-quantitative detection of six important curable sexually transmitted pathogens and therefore represents an alternative method for the clinical detection and monitoring of STIs.

Evaluation of a Probe-Based PCR-ELISA System for Simultaneous Semi Quantitative Detection and Genotyping of Human Cytomegalovirus (HCMV) Infection in Clinical Specimens.

BACKGROUND: Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance. METHOD: During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system. RESULTS: PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized conditions of hybridization, differentiation between genotypes of glycoprotein B was feasible using genotype-specific probes in PCR-ELISA genotyping system. In comparison with sequencing method, genotyping system was confirmed with kappa index of 1. CONCLUSION: PCR-ELISA is proposed as an applicable and reliable technique for semi-quantitative diagnosis and typing of the infection. This technique is flexible to apply in a variety of molecular fields.