Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity

Sequential method for rapid early diagnosis of white spot syndrome virus in crayfish

We developed a practical method to rapidly detect and diagnose latent white spot syndrome virus (WSSV) in infected crayfish that were non-symptomatic for WSSV. This method included a simplified extraction of DNA template, optimized loop-mediated isothermal amplification (LAMP), and final visualization of the product by means of staining with SYBR green I. Using this method, WSSV was detected in crayfish that had been artificially infected in two ways: at 5 h after injection, and 24 h after feeding with tissue from WSSV-infected crayfish (at a stage when such infected crayfish were non-symptomatic), and a thousand times or more dilution can omit fluorescent background when SYBR green I was used. Results indicate that this was a rapid, convenient, and highly sensitive method for the early diagnosis and detection of WSSV. The whole detection procedure took less than one hour to complete.Key words: White spot syndrome virus, loop-mediated isothermal amplification, SYBR green I, Procambarus clarkii, early diagnosis

Development of Sybr Green I-Based Melting Curve Method for HER2I655V Polymorphism Detection in Breast Cancer

Background: Currently available molecular method to detect HER2I655V polymorphism such as PCR-RFLP is hampered by the costly experimental method, and post-PCR treatment requirement that makes this technique is not meeting for high-throughput analysis purpose. In this study, we developed an accurate, simple, low cost and rapid test to detect polymorphism at HER2 gene using SBR Green I based-melting curve method. Methods: Two forward allele-specific primers and one common reverse primer were used then these primers were tested to discriminate known genotypes of genomic templates (GG type or AA type) and genomic samples retrieved from breast cancer patients. Results: Melting curve analysis derived from SYBR Green I-based allele-specific PCR with defined primers concentration and annealing temperature at 54.3 °C showed good discrimination level of Tm peaks in which GG genotype melted at 89 °C slightly higher than AA genotype which melted at 86 °C, while AG genotype harbored both of homozygous Tm characteristics. Conclusions: This preliminary result will be as basic for further large-scale typing of HER2I655V polymorphism.&nbsp

Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR

Background Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV). One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR) assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture. Results One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using in vitro transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R2) of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10-1 to 10-5 TCID50, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products. Conclusions The one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model

Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR

<p>Abstract</p> <p>Background</p> <p>Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV). One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR) assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture.</p> <p>Results</p> <p>One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using <it>in vitro </it>transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R²) of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10^-1to 10^-5TCID₅₀, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products.</p> <p>Conclusions</p> <p>The one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model.</p

Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria

A new nucleic acid stain, SYBR Green I, can be used for the rapid and accurate determination of viral and bacterial abundances in diverse marine samples. We tested this stain with formalin-preserved samples of coastal water and also from depth profiles (to 800 m) from sites 19 and 190 km offshore, by filtering a few ml onto 0.02 μn pore-size filters and staining for 15 min. Comparison of bacterial counts to those made with acridine orange (AO) and virus counts with those made by transmission electron microscopy (TEM) showed very strong correlations. Bacterial counts with AO and SYBR Green I were indistinguishable and almost perfectly correlated (r2 = 0.99). Virus counts ranged widely, from 0.03 to 15 × 107 virus ml-1. Virus counts by SYBR Green I were on the average higher than those made by TEM, and a SYBR Green I versus TEM plot yielded a regression slope of 1.28. The correlation between the two was very high with an r2 value of 0.98. The precision of the SYBR Green I method was the same as that for TEM, with coefficients of variation of 2.9%. SYBR Green I stained viruses and bacteria are intensely stained and easy to distinguish from other particles with both older and newer generation epifluorescence microscopes. Detritus is generally not stained, unlike when the alternative dye YoPro I is used, so this approach may be suitable for sediments. SYBR Green I stained samples need no desalting or heating, can be fixed with formalin prior to filtration, the optimal staining time is 15 min (resulting in a total preparation time of less than 25 min), and counts can be easily performed at sea immediately after sampling. This method may facilitate incorporation of viral research into most aquatic microbiology laboratories

Comparison of the SYBR Green and the hybridization probe format for real-time PCR detection of HHV-6

A comparative study was conducted of a novel real-time quantitative PCR test (LightCycler System) with FastStart DNA Master(PLUS) SYBR Green I dye to detect DNA of human herpes virus 6 (HHV-6). Results were compared with those of a real-time quantitative PCR with hybridization probe (HP) formats using the fluorescence resonance energy transfer method, and with those of a single qualitative PCR test. The detection limit of the test with SYBR Green I dye was 20 copies of the virus, similar to that of the other two tests. The reproducibility was satisfactory. The new test has the same advantages as real-time PCR with HP formats and offers a greater versatility at lower cost

Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Although a few alternative dyes without some of these limitations have been recently proposed, no large-scale investigation into the properties of intercalating dyes has been performed. In this study, we investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit PCR, show no preferential binding to GC rich sequences and do not influence melting temperature, Tm, even at high concentrations. In addition, SYTO-82 demonstrated a 50-fold lower detection limit in a dilution series assay. In conclusion, the properties of SYTO-82 and SYTO-13 will simplify the development of multiplex assays and increase the sensitivity of real-time PCR

Electrostatically Directed Visual Fluorescence Response of DNA-Functionalized Monolithic Hydrogels for Highly Sensitive Hg2+ Detection

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Applied Materials and Interfaces, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/am101068cHydrogels are cross-linked hydrophilic polymer networks with low optical background and high loading capacity for immobilization of biomolecules. Importantly, the property of hydrogel can be precisely controlled by changing the monomer composition. This feature, however, has not been investigated in the rational design of hydrogel-based optical sensors. We herein explore electrostatic interactions between an immobilized mercury binding DNA, a DNA staining dye (SYBR Green I), and the hydrogel backbone. A thymine-rich DNA was covalently functionalized within monolithic hydrogels containing a positive, neutral, or negative backbone. These hydrogels can be used as sensors for mercury detection since the DNA can selectively bind Hg2+ between thymine bases inducing a hairpin structure. SYBR Green I can then bind to the hairpin to emit green fluorescence. For the neutral or negatively charged gels, addition of the dye in the absence of Hg2+ resulted in intense yellow background fluorescence, which was attributed to SYBR Green I binding to the unfolded DNA. We found that, by introducing 20% positively charged allylamine monomer, the background fluorescence was significantly reduced. This was attributed to the repulsion between positively charged SYBR Green I by the gel matrix as well as the strong binding between the DNA and the gel backbone. The signal-to-background ratio and detection limit was, respectively, improved by 6- and 9-fold using the cationic gel instead of neutral polyacrylamide gel. This study helps understand the electrostatic interaction within hydrogels, showing that hydrogels can not only serve as a high capacity matrix for sensor immobilization but also can actively influence the interaction between involved molecules.University of Waterloo || Natural Sciences and Engineering Research Council |

Quantification of substance P mRNA expression in the midbrain of ovariectomized migraine rats with SYBR green I real-time polymerase chain reaction

This study was designed to develop a SYBR green I-based real-time polymerase chain reaction (RTPCR) for quantitative detection of substance P (SP) mRNA in the midbrain of ovariectomized migraine rats and to evaluate the effects of estradiol on the mRNA expression of SP in order to shed light on the mechanisms underlying the pathogenesis of migraine and estrogen-conferred protection against migraine. 24 female rats were randomly assigned to the following groups: A: non-migraine controls; B: migraines; C, migraine rats receiving low estradiol; D, migraine rats receiving high estradiol. One week following ovariectomy, migraine was induced in groups B, C and D by nitroglycerin (i.p.). Behavior changes before and after migraine was examined. A SYBR green I-based RT-PCR assay was established to measure the absolute levels of SP mRNA in the midbrain. Behavioral changes in group D were significantly mitigated when compared with those in group B, whereas no marked behavioral changes were noted in groups C and B. In addition, mRNA copies of SP in group B were remarkably lower than group A, while the level of SP mRNA in both groups C and D was higher than group B, although no significance was reached (P &gt; 0.05). SP mRNA expression decreased in the midbrain of migraine rats when compared with the non-migraine controls. High doses of estrogen partially restored SP expression in migraine rats and reduce migraine attack. Our study validated the SYBR green I-based RT-PCR technique for quantitative detection of SP mRNA.Keywords: Substance P, migraine, estrogen, midbrain, real-time quantitative polymerase chain reaction, ratsAfrican Journal of Biotechnology Vol. 9(34), pp. 5481-5487, 23 August, 201