A facile electrochemical aptasensor for chloramphenicol detection based on synergistically photosensitization enhanced by SYBR Green I and MoS2.

This study reports the development of a photocatalytic electrochemical aptasensor for the purpose of detecting chloramphenicol (CAP) antibiotic residues in water by utilizing SYBR Green I (SG) and chemically exfoliated MoS2 (ce-MoS2) as synergistically signal-amplification platforms. The Au nanoparticles (AuNPs) were electrodeposited onto the surface of an indium tin oxide (ITO) electrode. After that, the thiolate-modified cDNA, also known as capture DNA, was combined with the aptamer. Subsequently, photosensitized SG molecules and ce-MoS2 nanomaterial were inserted into the groove of the resultant double-stranded DNA (dsDNA). The activation of the photocatalytic process upon exposure to light resulted in the generation of singlet oxygen. The singlet oxygen effectively split the dsDNA, resulting in significant enhancement in the current of [Fe(CN)6]3-/4-. When the CAP was present, both SG molecules and ce-MoS2 broke away from the dsDNA, which turned off the photosensitization response, leading to significant reduction in the current of [Fe(CN)6]3-/4-. Under the optimal conditions, the aptasensor exhibited a linear relationship between the current of [Fe(CN)6]3-/4- with logarithmic concentrations of CAP from 20 to 1000 nM, with a detection of limit (3σ) of 3.391 nM. The aptasensor also demonstrated good selectivity towards CAP in the presence of interfering antibiotics, such as tetracycline, streptomycin, levofloxacin, ciprofloxacin, and sulfadimethoxine. Additionally, the results obtained from the analysis of natural water samples using the proposed aptasensor were consistent with the findings acquired through the use of a liquid chromatograph-mass spectrometer. Therefore, with its simplicity and high selectivity, this aptasensor can potentially detect alternative antibiotics in environmental water samples by replacing the aptamers based on photosensitization.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

A SYBR Green I-based aptasensor for the label-free, fluorometric, and anti-interference detection of MeHg.

Methylmercury (MeHg+) is a common form of organic mercury that is substantially more toxic than inorganic mercury and is more likely to accumulate in organisms through biological enrichment. Therefore, developing a method to enable the specific and rapid detection of MeHg+ in seafood is important and remains challenging to accomplish. Herein, a rapid, label-free fluorescence detection method for MeHg+ determination was developed based on SYBR Green I. The detection system implemented "add and measure" detection mode can be completed in 10 min. Under optimal assay conditions, the detection platform showed a linear relationship with the concentration of MeHg+ within 1-50 nM (Y = 8.573x + 42.89, R2 = 0.9928), with a detection limit of 0.3218 nM. The results obtained for competitive substances, such as inorganic mercury ions and anions, show a high specificity of the method. In addition, this method successfully detected MeHg+ in seawater and marine products, with an accompanying spike recovery rate of 96.45-105.1%.

Diagnosis of pleural tuberculosis by multi-targeted loop-mediated isothermal amplification assay based on SYBR Green I reaction: comparison with GeneXpert® MTB/RIF assay.

BACKGROUND: Diagnosis of pleural tuberculosis (TB) is tedious owing to its close resemblance with malignant pleural effusion and sparse bacterial load in clinical specimens. There is an immediate need to design a rapid and dependable diagnostic test to prevent unnecessary morbidity/mortality. RESEARCH DESIGN AND METHODS: A multi-targeted loop-mediated isothermal amplification (MT-LAMP) was deliberated using mpt64 and IS6110 to diagnose pleural TB within pleural fluids/biopsies. MT-LAMP products were analyzed by gel-based and visual detection methods, viz. SYBR Green I, SYBR Green I+deoxyuridine triphosphate uracil-N-glycosylase (dUTP-UNG), and dry methyl green reactions. RESULTS: In a pilot study, while assessing pleural TB/non-TB control subjects (n = 40), both SYBR Green I+dUTP-UNG/gel-based MT-LAMP assays exhibited better sensitivity/specificity than SYBR Green I and dry methyl green MT-LAMP. Since it is facile to work with SYBR Green I+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green I+dUTP-UNG in a higher number of specimens (n = 97), which revealed somewhat higher sensitivity (85.2 vs. 81.5%) and specificity (97.7 vs. 90.7%) than SYBR Green I MT-LAMP. Furthermore, the sensitivity attained by SYBR Green I+dUTP-UNG MT-LAMP was significantly higher (p < 0.001) than GeneXpert. CONCLUSIONS: Our SYBR Green I+dUTP-UNG MT-LAMP is a simple and reliable method to diagnose pleural TB, which may translate into a point-of-care test.

A recombinase polymerase amplification-SYBR Green I assay for the rapid and visual detection of Brucella.

Brucellosis is a zoonosis caused by Brucella, which poses a great threat to human health and animal husbandry. Pathogen surveillance is an important measure to prevent brucellosis, but the traditional method is time-consuming and not suitable for field applications. In this study, a recombinase polymerase amplification-SYBR Green I (RPAS) assay was developed for the rapid and visualized detection of Brucella in the field by targeting BCSP31 gene, a conserved marker. The method was highly specific without any cross-reactivity with other common bacteria and its detection limit was 2.14 × 104 CFU/mL or g of Brucella at 40 °C for 20 min. It obviates the need for costly instrumentation and exhibits robustness towards background interference in serum, meat, and milk samples. In summary, the RPAS assay is a rapid, visually intuitive, and user-friendly detection that is highly suitable for use in resource-limited settings. Its simplicity and ease of use enable swift on-site detection of Brucella, thereby facilitating timely implementation of preventive measures.

Comparison of SYBR green I and lactate dehydrogenase antimalarial in vitro assay in Plasmodium falciparum field isolates.

Several assay methods are in use for monitoring the drug sensitivity of malaria parasites and screening new antimalarial drugs. Plasmodium lactate dehydrogenase (pLDH) and SYBR Green I in vitro assays were used to evaluate the drug efficacy of Chloroquine, Artemisinin and Azadirachta indica silver nano particles against Plasmodium falciparum 3D7 strain. The half-maximal inhibitory concentration (IC50) of each compound was estimated with non-linear regression model - dose-response analysis. The consistency between two methods was analysed with Cohen's kappa coefficient, interclass correlation and Bland-Altman plots. No statistical difference was found between IC50 values determined by both assays (p = 0.714). The proportion of resistant isolates to chloroquine according to SYBR green I (43.48%) and pLDH (34.78%) assays were similar (z = 0.302; p = 0.762) with significant concordant between methods (k = 0.819, p < 0.001). The results of pLDH Qualisa assay was comparable with classic SYBR green I assay and can be potentially useful in antimalarial drug efficacy surveillance.

A Multiplex PCR Melting-Curve-Analysis-Based Detection Method for the Discrimination of Five Aspergillus Species.

Aspergillus mold is a ubiquitously found, airborne pathogen that can cause a variety of diseases from mild to life-threatening in severity. Limitations in diagnostic methods combined with anti-fungal resistance render Aspergillus a global emerging pathogen. In industry, Aspergilli produce toxins, such as aflatoxins, which can cause food spoilage and pose public health risk issues. Here, we report a multiplex qPCR method for the detection and identification of the five most common pathogenic Aspergillus species, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans. Our approach exploits species-specific nucleotide polymorphisms within their ITS genomic regions. This novel assay combines multiplex single-color real time qPCR and melting curve analysis and provides a straight-forward, rapid, and cost-effective detection method that can identify five Aspergillus species simultaneously in a single reaction using only six unlabeled primers. Due to their unique fragment lengths, the resulting amplicons are directly linked to certain Aspergillus species like fingerprints, following either electrophoresis or melting curve analysis. Our method is characterized by high analytical sensitivity and specificity, so it may serve as a useful and inexpensive tool for Aspergillus diagnostic applications both in health care and the food industry.

Gene Expression Analysis by Quantitative Real-Time PCR for Floral Tissues.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR) is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental set-up used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler® 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment.

Optical Polymorphism of Liquid-Crystalline Dispersions of DNA at High Concentrations of Crowding Polymer.

Optically active liquid-crystalline dispersions (LCD) of nucleic acids, obtained by polymer- and salt-induced (psi-) condensation, e.g., by mixing of aqueous saline solutions of low molecular weight DNA (≤106 Da) and polyethylene glycol (PEG), possess an outstanding circular dichroism (CD) signal (so-called psi-CD) and are of interest for sensor applications. Typically, such CD signals are observed in PEG content from ≈12.5% to ≈22%. However, in the literature, there are very conflicting data on the existence of psi-CD in DNA LCDs at a higher content of crowding polymer up to 30-40%. In the present work, we demonstrate that, in the range of PEG content in the system above ≈24%, optically polymorphic LCDs can be formed, characterized by both negative and positive psi-CD signals, as well as by ones rather slightly differing from the spectrum of isotropic DNA solution. Such a change in the CD signal is determined by the concentration of the stock solution of PEG used for the preparation of LCDs. We assume that various saturation of polymer chains with water molecules may affect the amount of active water, which in turn leads to a change in the hydration of DNA molecules and their transition from B-form to Z-form.

Quantitative PCR of Alu Repeats Using PowerUp™ SYBR® Green Master Mix.

Quantitative PCR is one of the fundamental steps performed when processing routine casework in a forensic laboratory. Quantitative PCR of Alu repeats using a SYBR® Green master mix can produce calculated estimates of how much DNA was extracted from a sample. This process offers more efficiency, human specificity, and can be performed faster than other outdated quantification methods, such as slot blot or yield gel. A qPCR master mix is prepared and consists of Alu-F primers, Alu-R primers, water, and SYBR® Green master mix. The Alu-F and Alu-R primers target Alu sequences that are present hundreds of thousands of times throughout the human genome and are effective markers for human DNA quantification. During qPCR, the 7500 system facilitates the amplification of target Alu repeats. The SYBR® Green I fluorescent dye intercalates between the amplified dsDNA targets. During each amplification cycle, the 7500 system agitates the SYBR® Green I dye, resulting in a fluorescence signal that is recorded when it passes a specified Ct value. After qPCR amplification is complete, a standard curve is created and used to determine how much DNA a sample contains. This chapter provides instructions on how to accurately prepare a 96-well plate for qPCR, use the 7500 system and associated software to set up the qPCR amplification, and interpret the corresponding results produced.

Novel rapid detection of melamine based on the synergistic aggregation of gold nanoparticles.

A simple and rapid colorimetric method for the detection of melamine in milk samples is described. Polythymidine oligonucleotide was adsorbed on to the surface of gold nanoparticles (AuNPs), protecting it from aggregation. In the presence of melamine, polythymidine oligonucleotide combined with melamine formed a double-strand DNA-like structure, allowing AuNPs aggregation. In the presence of positively charged SYBR Green I (SG I), AuNPs were further aggregated. In the presence of melamine and SG I, aggregation of AuNPs was synergistic. Thus, in this principle, melamine can be detected visually. Plasmon resonance peak changes enabled detection of melamine quantitatively using UV-vis spectroscopy. The limit of detection for this colorimetric method was 16 µg L-1 with a good linear range from 19.5 µg L-1 to 1.25 × 103 µg L-1, and detection took only 1 min. The method was successfully applied for detection of melamine in milk samples.

Staining Properties of Selected Commercial Fluorescent Dyes Toward B- and Z-DNA.

The properties of six commonly used, commercially available, fluorescent dyes were compared in staining right-handed B-DNA and left-handed Z-DNA. All showed different degree of fluorescence turn-on in the presence of B-DNA, but very little in the presence of Z-DNA. The optimal range of dye-DNA ratios of DNA was determined. While these dyes do not provide a turn-on type probe for Z-DNA, staining between B- and Z-DNA using dyes such as SYBR Green I was shown to be useful in tracking the kinetics of conformational changes between these two forms of DNA. Finally, SYBR Green I showed unique circular dichroism patterns in 4 M NaCl that change in the presence of double stranded DNA, both in the visible and UV range.

Distinction of Male and Female Trees of Ginkgo biloba Using LAMP.

Ginkgo biloba is utilized as food, medicine, wood, and street trees among other things. The objective of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for gender distinction of G. biloba. Male-specific SCAR gene can be utilized to identify G. biloba gender using LAMP. The optimized LAMP conditions, temperature 60 °C, 2-mM MgSO4, and [F3/B3]:[FIP/BIP] primer ratio of 1:4 were selected as final conditions. The G. biloba SCAR LAMP displayed a sensitivity of 10 ng when amplified by concentration under the optimum conditions. Additionally, it demonstrated a particular response in male with SYBR Green I in LAMP analysis that can be a more powerful tool for field and scale-up applications. Our work represents a first attempt to identify G. biloba gender using LAMP and offers an efficient and reliable tool for roadside landscaping.

A tag-free fluorescent aptasensor for tobramycin detection using a hybridization of three aptamer strands and SYBR Green I dye.

In this study, a sensitive fluorescent method is designed to detect tobramycin (TOB) drug applying a hybrid structure of three aptamer strands and SYBR Green I (SGI) fluorescent dye as the bioreceptor segment and signal indicator, respectively. The preferential binding of the aptamers to TOB resulted in the collapse of the hybridized aptamer skeleton to the single strands. So, the intercalation of SGI molecules reduced that quenched the fluorescence response. The aptasensing assay provided the superior target specificity with a detection limit (LOD) of 0.153 pM and a wide linear dynamic range over 0.5 pM-300 µM. The aptasensor could successfully quantify TOB in human serum samples. The tag-free sensor with the remarkable advantages of simplicity, easy-to-use, cost-effectiveness, and high sensitivity is superior to be applicable for clinical samples.

A Novel Fluorescent Sensor Based on Aptamer and qPCR for Determination of Glyphosate in Tap Water.

Glyphosate (GLYP) is a broad-spectrum, nonselective, organic phosphine postemergence herbicide registered for many food and nonfood fields. Herein, we developed a biosensor (Mbs@dsDNA) based on carboxylated modified magnetic beads incubated with NH2-polyA and then hybridized with polyT-glyphosate aptamer and complementary DNA. Afterwards, a quantitative detection method based on qPCR was established. When the glyphosate aptamer on Mbs@dsDNA specifically recognizes glyphosate, complementary DNA is released and then enters the qPCR signal amplification process. The linear range of the method was 0.6 µmol/L−30 mmol/L and the detection limit was set at 0.6 µmol/L. The recoveries in tap water ranged from 103.4 to 104.9% and the relative standard deviations (RSDs) were <1%. The aptamer proposed in this study has good potential for recognizing glyphosate. The detection method combined with qPCR might have good application prospects in detecting and supervising other pesticide residues.

Measurement of Babesia bovis infected red blood cells using flow cytometry.

Rapid and accurate tools are needed for high-throughput in vitro antibabesial drug testing. In this study, flow cytometry for the measuring of Babesia bovis in vitro culture, was developed using SYBR Green I and compared against the results of fluorescence-based assay and microscopic assay. A high correlation of measured parasitemia was observed with high R2 value (R2 = 0.9991) between flow cytometry and microscopic analysis. The degree of antibabesial drug sensitivity against B. bovis determined by flow cytometry was 0.424 ± 0.173 µM. Similar to the results of previously published studies involving fluorescence spectrometry-based assay (0.408 ± 0.011 µM) and microscopy-based assay (0.400 ± 0.017 µM). The outcomes of this present study suggest that flow cytometry assay using SYBR Green I can potentially be useful in determining parasitemia and can serve as a rapid alternative method to antibabesial drug testing.

Enhanced resolution of marine viruses with violet side scatter.

Marine viruses make up an essential compartment of the marine ecosystem. They are the most abundant organisms and represent one of the biggest sources of unknown biodiversity. Viruses also have an important impact on bacterial and algal mortality in the ocean, and as such have a major influence on microbial diversity and biogeochemical cycling. However, little is known about the abundance and distribution patterns of viruses across the oceans and seas. Over the last 20 years, flow cytometry has been the technique of choice to detect and count the viral particles in natural samples. Nevertheless, due to their small size, the detection of marine viruses is still extremely challenging. In this article we describe how a new generation of flow cytometer which uses the side scatter (SSC) of violet photons from a 405 nm laser beam helps to improve the resolution for detecting marine viruses. To the best of our knowledge, this is the first report where virioplankton has been detected in aquatic samples using flow cytometry with a 405 nm violet SSC instead of a 488 nm blue SSC.

Rapid visual detection of anisakid nematodes using recombinase polymerase amplification and SYBR Green I.

Anisakidosis is a food-borne parasitic disease (FBPD) caused by the third-stage larvae of the family Anisakidae. Therefore, it is important to develop a simple, rapid and equipment-free detection method for anisakids in fish samples or seafood since current methods are time-consuming and require complex instruments. In this study, a recombinase polymerase amplification (RPA)-based method was established for the first time to detect anisakids by targeting the internal transcribed spacer (ITS) regions. The detection results were visualized by including SYBR Green I (SG) in the method. The sensitivity of RPA-SG assay was 102 copies per reaction of recombinant plasmid (within 20 min at 37°C), similar to quantitative real-time PCR (qPCR). The assay had high specificity for detecting anisakids against other related parasites and host fish. In addition, the assay was further used to detect fresh marine fish contaminated with anisakids and it showed high precision. These results indicate that the novel RPA-SG assay suitable for visual detection of anisakids in the field and food safety control.

Detection of touch DNA evidence on swab by SYBR®Green I Nucleic Acid Gel Stain.

Invisible touch DNA sample is one of the most common sample types received in the DNA laboratory. However, the appearance of DNA from touch sample collection cannot be observed until the DNA detection process is completed. The objective of this study focused on effectiveness of SYBR®Green I Nucleic Acid Gel Stain (SYBR Green I, Invitrogen, USA) using four types of swabs; including cotton, rayon, nylon, and foam swab, for collecting touch DNA from objects. Touch DNA samples were collected from 1 male and 1 female volunteers and stained with 5x SYBR Green I. This study demonstrated 3 experimental designs; including effectiveness, stability and visualizing of touch DNA mockup study. The results showed that the foam swab was the only swab type that could distinguish between touch DNA with and without SYBR Green I fluorescence; therefore, the foam swab was studied in the mockup experiment step and was appropriate for the study of the stability of SYBR Green I stained swab. Furthermore, our results on stability study showed that touch DNA on foam swab stained with 5x SYBR Green I fluorescence immediately faded on Day 1 , but remained until Day 28 in the dark at room temperature. The mockup study using a foam swab that collected touch DNA on objects and stained it with 5x SYBR Green I revealed that SYBR Green I fluorescence was visualized differently from that without touch DNA. These outcomes indicated that SYBR Green I can be used as a stain in the touch DNA screening method on foam swabs for forensic laboratory, which would increase productivity by reducing time and resources cost.

Diagnosis of abdominal tuberculosis by multi-targeted (mpt64 and IS6110) loop-mediated isothermal amplification assay.

BACKGROUND AND AIM: Diagnosis of abdominal TB is an exigent task due to variable anatomical sites and non-specific clinical manifestations that closely resemble other diseases. Most of the available diagnostic modalities yield low sensitivities and need expertise to handle the specialized equipment. Hence, there is an urgent need to develop a rapid and reliable diagnostic test, so as to reduce the unnecessary morbidity. Therefore, we designed a multi-targeted loop-mediated isothermal amplification (MT-LAMP) for diagnosing abdominal TB. METHODS: We evaluated an MT-LAMP (using mpt64 and IS6110) to diagnose abdominal TB within ascitic fluids and intestinal/peritoneal biopsies and compared these results with multiplex-PCR (M-PCR) using the same targets. MT-LAMP products were analyzed by gel electrophoresis and visual detection methods, that is, hydroxy naphthol blue and SYBR Green I reaction. RESULTS: Sensitivities of 80.9% and 84.6% were obtained in suspected (n = 42) and total abdominal TB (n = 52) cases, respectively by gel-based MT-LAMP, with 97.3% (n = 37) specificity in non-TB controls. Notably, sensitivities attained by gel-based/SYBR Green I MT-LAMP in both clinically suspected and total abdominal TB cases were significantly higher (P < 0.05) than M-PCR. Furthermore, sensitivity obtained with SYBR Green I was equivalent to that of gel-based MT-LAMP, while somewhat lesser specificity (94.6%) was attained with SYBR Green I, compared with gel-based MT-LAMP. CONCLUSION: Both gel-based and SYBR Green MT-LAMP exhibited equivalent sensitivities to diagnose abdominal TB. Because SYBR Green LAMP is easier to perform than a gel-based assay, we are currently focused on improving the specificity of this assay so as to develop a diagnostic kit.