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%.

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.

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.

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.

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.

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.

Two-step aggregation of gold nanoparticles based on charge neutralization for detection of melamine by colorimetric and surface-enhanced Raman spectroscopy platform.

A colorimetric and surface-enhanced Raman scattering (SERS) signal amplification platform based on 2-step aggregation of gold nanoparticles (AuNP) was constructed for the sensitive detection of melamine. In this study, the positively charged SYBR Green I was used for the first step of aggregation of AuNP, via charge neutralization, to obtain small-sized AuNP aggregates. The positively charged SYBR Green I decreased the negative charges of the surface of AuNP, which was beneficial to the aggregation of AuNP. In addition, the melamine could aggregate AuNP by decreasing the negative charges of the surface of AuNP and self-assemble with each other on the surface of AuNP by hydrogen bonds. Therefore, the second efficient aggregation of small-sized AuNP aggregates could be achieved with melamine at low concentration, resulting in significant signal changes of color and SERS. The sensitivity of a colorimetric (0.60 mg/L) and SERS (0.089 mg/L) platform, based on 2-step aggregation of AuNP, was 15 and 2.2 times higher than that based on 1-step aggregation of AuNP for detecting melamine.

RPA-SYBR Green I based instrument-free visual detection for pathogenic Yersinia enterocolitica in meat.

Pathogenic Yersinia (Y.) enterocolitica is the primary causative agent of Yersiniosis, with outbreaks in numerous countries around the world, and causes diarrhea and vomiting in animals and humans. Therefore, an instrument-free and convenient nucleic acid visualization method, RPA-SYBR Green I, was established, which combines recombinase polymerase amplification (RPA) with the fluorescent dye SYBR Green I for the detection of the adhesion gene ail in pathogenic Y. enterocolitica. After optimization of a series of conditions such as primer concentration, the detection of pathogenic Y. enterocolitica could be finally completed within about 20 min (from DNA extraction to observation of results) at an isothermal temperature of 39°C. RPA-SYBR Green I had no cross-reactivity with other bacteria and the detection limit was 101 CFU/µL, with sensitivity equal to that of conventional PCR. The method established in this paper and conventional PCR identified a total of 5 spiked samples and 15 meat samples stored in refrigerated, and it was concluded that there was 100% consistency between the two methods. Overall, RPA-SYBR Green I is a visual and facilitate detection assay that can accurately discover pathogenic Y. enterocolitica.

Exploring confocal laser scanning microscopy (CLSM) and fluorescence staining as a tool for imaging and quantifying traces of marine microbioerosion and their trace-making microendoliths.

Microscopic organisms that penetrate calcareous structures by actively dissolving the carbonate matrix, namely microendoliths, have an important influence on the breakdown of marine carbonates. The study of these microorganisms and the bioerosion traces they produce is crucial for understanding the impact of their bioeroding activity on the carbonate recycling in environments under global climate change. Traditionally, either the extracted microendoliths were studied by conventional microscopy or their traces were investigated using scanning electron microscopy (SEM) of epoxy resin casts. A visualisation of the microendoliths in situ, that is within their complex microbioerosion structures, was previously limited to the laborious and time-consuming double-inclusion cast-embedding technique. Here, we assess the applicability of various fluorescence staining methods in combination with confocal laser scanning microscopy (CLSM) for the study of fungal microendoliths in situ in partly translucent mollusc shells. Among the tested methods, specific staining with dyes against the DNA (nuclei) of the trace making organisms turned out to be a useful and reproducible approach. Bright and clearly delineated fluorescence signals of microendolithic nuclei allow, for instance, a differentiation between abandoned and still populated microborings. Furthermore, infiltrating the microborings with fluorescently stained resin seems to be of great capability for the visualisation and quantification of microbioerosion structures in their original spatial orientation. Potential fields of application are rapid assessments of endolithic bio- and ichnodiversity and the quantification of the impact of microendoliths on the overall calcium carbonate turnover. The method can be applied after CLSM of the stained microendoliths and retains the opportunity for a subsequent investigation of epoxy casts with SEM. This allows a three-fold approach in studying microendoliths in the context of their microborings, thereby fostering the integration of biological and ichnological aspects of microbial bioerosion.

Bioerosion describes the process of active erosion of hard substrates induced by the activity of living organisms. Beside numerous marine macroscopic bioeroding organisms such as sponges, annelids or bivalves, there is an astonishing 'hidden diversity' of microscopic bioeroding organisms which produce minute tunnels and chambers, for example in calcareous shells and skeletons of other marine organisms. These so-called microendoliths belong to bacteria, microalgae, foraminiferans, or fungi. Due to their lifestyle hidden inside the hard substrate, scientific investigation is often laborious and involves complex preparation techniques, electron microscopy, or even nano-computed tomography. Photo-autotrophic microendoliths (eg cyanobacteria and algae) have been studied with fluorescence microscopy using autofluorescence properties, for example of their chloroplasts. However, microendoliths of aphotic depths, mostly of fungal origin, do not show autofluorescence. With the present study we test different fluorescent dyes staining the microbioeroders 'in situ', that is, inside their microscopic tunnels, and visualise them using three-dimensional confocal laser scanning microscopy (CLSM). Very good results have been obtained with the dye Sybr Green I that stains DNA molecules and thereby the cell nuclei of the microendoliths. This method can be used, for instance, to measure the infestation rate of a given substrate by discriminating between abandoned microborings and those still inhabited by microendoliths. Another approach that was successfully tested in the course of the present study was the infiltration of the cleaned microborings with resin that was previously mixed with the fluorescent dye Safranin-O. The datasets obtained with the CLSM were used to reconstruct 3D-surface models of the microborings of three different microendoliths. Such models can be used to analyse the original spatial arrangement inside the hard substrate and to measure exact volumes. The resulting possibility to make exact quantifications is of high value for future investigations that focus on the role and proportion of microbioerosion in the (re)cycling of marine carbonates.

Validation of SYBR green I based closed-tube loop-mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria.

BACKGROUND: As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. METHODS: A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. RESULTS: LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. CONCLUSIONS: These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

A SYBR Green I-based real-time polymerase chain reaction assay for detection and quantification of canine bufavirus.

Canine bufavirus (CBuV) was first discovered in puppies in Italy in 2016, and subsequent studies have reported its possible relationship with acute enteritis. Currently, there is no specific and quantitative detection method for CBuV. This study examined the conserved NS1 gene and used a pair of specific primers to establish a direct SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) method for the detection and quantification of CBuV. In the sensitivity experiment, the detection limit of SYBR Green I-based real-time qPCR was 4.676 × 101 copies/µL and that of conventional PCR (cPCR) was 4.676 × 103 copies/µL. Furthermore, the qPCR method did not detect other viruses in dogs, indicating good specificity. The intra-assay coefficient of variation was 0.07-0.55% and the inter-assay coefficient of variation was 0.03-0.11%, indicating good repeatability. In clinical sample testing, the detection rate of qPCR was 5.0% (6/120), higher than that of cPCR (2.5%, 3/120). In addition, the samples that tested CBuV-positive in this experiment were all from dogs with acute enteritis. In summary, the SYBR Green I-based qPCR method established in this study has good sensitivity, specificity, and reproducibility for clinical sample detection and can also assist in future research on CBuV.

Development and application of SYBR Green â real-time quantitative reverse transcription PCR assay for detection of swine Getah virus.

Getah virus (GETV), a mosquito-borne virus belonging to the Alphavirus genus of family Togaviridae, has become increasingly problematic, which poses a huge threat to the safety of animals and public health. In order to detect GETV quickly and accurately, we have developed a SYBR Green I real-time quantitative reverse transcription PCR (RT-qPCR) assay for GETV with the detection limit of 66 copies/µL, excellent correlation coefficient (R2) of 0.9975, and amplification efficiency (E) of 98.90%, the target selected was the non-structural protein 3 of GETV. The sensitivity of it was higher than that of ordinary RT-PCR by 1000 folds, and the inter-assay and intra-assay CV values were all less than 0.99%. The newly developed RT-qPCR assay exhibited good sensitivity and reproducibility, which will provide technical support for the reliable and specific rapid diagnosis, and quantitative analysis of GETV infection.

A label-free ratiometric method to detect Hg2+ based on structural change of DNA.

In this work, a simple ratiometric method has been designed to detect Hg2+ based on the structural change between double-stranded DNA (dsDNA) and its G-quadruplex structure. When Hg2+ was added, the designed G-quadruplex structure could change into the corresponding dsDNA by forming the T-Hg2+ -T mismatch. This kind of variation resulted in a decrease in the fluorescence of the G-quadruplex/N-methyl mesoporphyrin IX (NMM) complex and an increase in the fluorescence from the dsDNA/SYBR Green I (SG I) pair. The secondary excitation wavelength of SG I was used to excite NMM and SG I simultaneously. The titration experiment indicated that the new method had a linear response within 0.7-2.5 µM Hg2+ with a limit of detection of 9.3 nM. Because using the T-Hg2+ -T mismatch to recognize Hg2+ was very specific, the selectivity of the new method was also satisfactory. The recoveries ranged from 92.8% to 110.2% suggested that this new method could achieve a potential application for Hg2+ detection in real environmental samples.

Development of loop-mediated isothermal method and comparison with conventional PCR assay for rapid on spot identification of tissue of cattle origin.

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for meat speciation with rapid and minimal equipment requirements. In this study, we developed cattle-specific tube-based LAMP assays targeting mitochondrial Cyt b gene sequence, compared with conventional PCR assay for specificity, sensitivity, and validation of the assay was made. The LAMP reaction was carried at 64 °C for 45 min, and results were confirmed by SYBR Green I dye and agarose gel-electrophoresis. The specificity of the assays was cross-tested with DNA of buffalo, goat, sheep, and pork. The amplification was observed with samples from cattle only without cross-reactivity with other meat species. The analytical sensitivity of LAMP and PCR method for cattle DNA detection was 0.0001 ng and 1 ng, respectively. Repeatability of the assay was achieved on samples from known/blind and admixture meat with other than cattle at the relative percentage of 20%, 10%, 5%, and 1%. The study concluded that the developed assay can be easily employed for the rapid identification of tissue of cattle origin in meat and meat products in low resource areas.

Simultaneous differentiation and diagnosis of goose parvovirus and astrovirus in clinical samples with duplex SYBR Green I real-time PCR.

Two pairs of primers were designed to bind conserved genomic regions of goose parvovirus (GPV) and goose astrovirus (GAstV) to establish a simple, sensitive, and highly specific duplex quantitative PCR (qPCR) method to simultaneously detect the two viruses. The duplex qPCR can distinguish GPV (melting point: 82.1 °C) and GAstV (melting point: 79.8 °C) by the peaks of their individual melting curves. Mixed testing with other waterfowl viruses produced no nonspecific peaks. The established standard curves showed good linear relationships (R2 > 0.997) and the limits of detection (LOD) for GPV and GAstV were 5.74 × 101 and 6.58 × 101 copies/µL, respectively. Both intra- and inter-assay coefficients of variation were <2%, indicating that the method has good repeatability. Twenty tissue samples from diseased geese were examined with the duplex qPCR assay and conventional PCR. Duplex qPCR showed positive rates of 25% for GPV and 45% for GAstV, and the positive rate for GPV and GAstV coinfection was 15%, slightly higher than the results for conventional PCR. These results indicated that this duplex qPCR method is highly sensitive, specific, and reproducible, and is suitable for epidemiological studies to effectively control the transmission of GPV and GAstV.

Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus.

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 101 copies/µL and 6.15 × 101 copies/µL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs.

Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 × 101 copies/µL, which was significantly more sensitive than that by gel electrophoresis of 6.4 × 103 copes/µL. This assay was specific and had no cross-reaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.

In Vitro Antimalarial and Toxicological Activities of Quercus infectoria (Olivier) Gall Extracts.

BACKGROUND: The spread of Plasmodium falciparum resistance in common antimalarial drugs, including artemisinin-based combination therapies, has necessitated the discovery of new drugs with novel mechanisms of action. In the present study, the in vitro antimalarial and toxicological activities of acetone, methanol, ethanol and aqueous extracts of Quercus infectoria (Q. infectoria) galls were investigated. METHODS: The extracts were assessed for the antimalarial potential using a malarial SYBR Green I fluorescence-based (MSF) assay, while the toxicity was screened by using brine shrimp lethality test (BSLT), haemolytic assay, and cytotoxicity assay against normal embryo fibroblast cell line (NIH/3T3) and normal kidney epithelial cell line (Vero). RESULTS: The acetone extract showed the highest antimalarial activity (50% inhibitory concentration, IC50 = 5.85 ± 1.64 µg/mL), followed by the methanol extract (IC50 = 10.31 ± 1.90 µg/mL). Meanwhile, the ethanol and aqueous extracts displayed low antimalarial activity with IC50 values of 20.00 ± 1.57 and 30.95 µg/mL ± 1.27 µg/mL, respectively. The significant antimalarial activity was demonstrated in all extracts and artemisinin (P < 0.05). All extracts were non-toxic to brine shrimps (50% lethality concentration, LC50 > 1000 ppm). Furthermore, no occurrence of haemolysis (< 5%) was observed in normal erythrocytes when treated with all extracts compared to Triton X-100 that caused 100% haemolysis (P < 0.05). The acetone and methanol extracts were non-toxic to the normal cell lines and statistically significant to artemisinin (P < 0.05). CONCLUSION: Taken together with satisfactory selectivity index (SI) values, the acetone and methanol extracts of Q. infectoria galls could serve as an alternative, promising and safe antimalarial agents.

Enumeration of the Invasion Efficiency of Plasmodium falciparum In Vitro in Four Different Red Blood Cell Populations Using a Three-Color Flow Cytometry-Based Method.

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum-infected RBCs and non-infected RBCs at 0.5% parasitemia were first labeled with 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) followed by co-culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co-cultures were harvested and stained with streptavidin-phycoerythrin (SA-PE) followed by staining of parasite-infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid-dependent) or 3D7 (sialic acid-independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme-treated RBC populations when compared with non-treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin-labeled RBCs will therefore provide a better understanding of invasion phenotype-specific host-parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry.

Development of a duplex SYBR Green I-based quantitative real-time PCR assay for the rapid differentiation of goose and Muscovy duck parvoviruses.

BACKGROUND: Waterfowl parvoviruses, including goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), can cause seriously diseases in geese and ducks. Developing a fast and precise diagnosis assay for these two parvoviruses is particularly important. RESULTS: A duplex SYBR Green I-based quantitative real-time PCR assay was developed for the simultaneous detection and differentiation of GPV and MDPV. The assay yielded melting curves with specific single peak (Tm = 87.3 ± 0.26 °C or Tm = 85.4 ± 0.23 °C) when GPV or MDPV was evaluated, respectively. When both parvoviruses were assessed in one reaction, melting curves with specific double peaks were yielded. CONCLUSION: This duplex quantitative RT-PCR can be used to rapid identify of GPV and MDPV in field cases and artificial trials, which make it a powerful tool for diagnosing, preventing and controlling waterfowl parvovirus infections.