scholarly journals Recruitment of cyanobacteria by reverse transcription quantitative real-time PCR based on expression of Microcystis gene

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7188
Author(s):  
Long Yu ◽  
Xiaofei Wu ◽  
Yang Yu ◽  
Limei Shi ◽  
Min Zhang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lake Taihu ◽  
Rna Extraction ◽  
Sybr Green ◽  
Quantitative Detection ◽  
Control Group ◽  
Quantitative Real Time Pcr ◽  
Total Rna ◽  
Pcr Method

In this study, a SYBR Green quantitative real-time PCR method was established and applied. Relative expression of the synthetic genes from Microcystis gas vesicles (gvpC), algal toxin genes (mcyA), and polysaccharides (espL) from water and sediments of Meiliang Bay and from the center of Lake Taihu were tested from January to June, 2017. Indoor Microcystis aeruginosa was used as the control group. The kit for total RNA extraction in Microcystis was optimized. Results showed that the optimized kit extracted high-concentrations and high-quality total RNA from Microcystis. The extraction purity and concentration were significantly higher than those extracted by the original kit. The transcription level of gvpC increased gradually until a peak was reached in March. However, expression of gvpC decreased continuously at the proliferating and floating stages of Cyanobacterial biomass. The maximum level of expression of gvpC in April in comparison to expression of mcyA in March occurred first. We found that the SYBR Green qRT-PCR method, which is characterized by high specificity, repeatability, is rapid, and can be used for quantitative detection of expression of gvpC, mcyA, and espL. The recruitment of cyanobacteria is the process in which cyanobacteria in the sediment began to regain their activity, started to grow and migrated to the water column.

Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
Author(s):  
ANNA-CLARA RÖNNER ◽  
HANS LINDMARK
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Close Correlation ◽  
Quantitative Detection ◽  
Quantitative Real Time Pcr ◽  
Direct Plating ◽  
Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

scholarly journals P0069MICRO RNA 155, 210, 494, 29B AND 34A EXPRESSION PROFILE IN PREECLAMPSIA AND NORMAL PREGNANCIES

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Ali Ghafari ◽  
Mahboob Lessan pezeshki ◽  
Mojtaba Saffari
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Consensus Sequence ◽  
Rna Extraction ◽  
Micro Rna ◽  
Control Group ◽  
Transcriptional Level ◽  
Total Rna ◽  
Qrt Pcr ◽  
Micro Rnas

Abstract Background and Aims Preeclampsia (PE) is a complex disorder that is characterized by hypertension and proteinuria after the 20th week of pregnancy, and it causes most neonatal morbidity and perinatal mortality. Despite many efforts, the knowledge acquired regarding its pathogenesis and pathophysiology does not allow us to treat it efficiently. It is not possible to arrest its progressive nature, and the available therapies are limited to symptomatic treatment. MicroRNAs (miRNAs) are small non-coding RNAs of approximately 19–23 nucleotides that can bind to the 3’ untranslated region of target mRNAs resulting in the degradation and translation inhibition of the mRNA, thereby regulating gene expression at the post-transcriptional level. Many studies have confirmed deregulated miRNA in pregnant patients with PE, and the function and mechanism of these differentially expressed miRNA are gradually being revealed. Method Sample collection Among the patients in the maternity ward, Department of Obstetrics and Gynecology. pregnant women who carried fetuses from 26 to 40 weeks of gestation were selected for study. Plasma samples were obtained from a total of 90 women in two groups; 48 being preeclamptic and 42 being healthy pregnancies, forming the control group. None of these subjects had undergone an invasive procedure. A volume of 5 mL maternal venous blood was drawn and collected in an ethylenediamine tetraacetic acid (EDTA) tube. The blood samples were centrifuged initially at 1200 g for 10 min and a second time at 10,000 g for 10 min, and 500 mL from the supernatant layer were used. The supernatant layer of the plasma was taken to Department of Molecular Biology and Genetics, Molecular Diagnosis Laboratory for storage at -80°C until the time of study. Total RNA extraction Total RNA was isolated from plasma using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer protocol. Total RNA was analyzed using the commercially available Bioanalyzer Agilent RNA 6000 picoassay. MicroRNA isolation from maternal plasma MicroRNAs were obtained with the High-Specificity miRNA QRT-PCR detection kit (Stratagene-an Agilent Technologies Company, USA-Canada) according to manufacturer recommendations. Micro RNAs were subjected to a polyadenylation reaction as previously described. Next, using an oligo dT primer harboring a consensus sequence, reverse transcription was performed using an Affinity Script RT/RNase Block Enzyme mixture (Stratagene). miRNA was anayzed using the Bioanalyzer 2100- Agilent Small RNA assay. QRT-PCR platform The cDNA was amplified by real-time PCR. The real-time PCR analysis was performed on a Stratagene Mx3005P. This reaction contained a microRNA-specific forward primer, a TaqMan probe complementary to the 3ꞌ of the specific microRNA sequence, as well as part of the polyA adaptor sequence, and a universal reverse primer complementary to the consensus 3ꞌ sequence of the oligodT tail. Results were recorded in clinic sheets and were analyzed using SPss version 22. Results G1 consist of 48 and G2 42 pts. There were no differences in two group in respect to age (mean age was 28± 9.5 and 27± 5.23 in G1 and G2 respectively). Micro RNA155, 210 and 494 were significantly higher in plasma of G1 (Pv 0.001, 0.0001, 0.005 respectively). Micro RNA 29b was significantly lower in G1 (Pv 003). And there was no difference between two groups in respect to Micro RNA 34a (Pv 0.09). And also PE patients with higher plasma creatinine have more plasma level of Micro RNA155 and 210. There was a significant correlation between Micro RNA 494 and anemia in PE patients Conclusion In this study we showed that Micro RNA155, 210 and 494 increased and Micro RNA 29b decreased in PE and after clarifying by further studies may use as a predictor in clinical practice.

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

A Quantitative Real-Time PCR Method Using an X-Linked Gene for Sex Typing in Pigs

2012 ◽  
Vol 54 (2) ◽  
pp. 493-496 ◽  
Author(s):  
Maria Ballester ◽  
Anna Castelló ◽  
Yuliaxis Ramayo-Caldas ◽  
Josep M. Folch
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Sex Typing ◽  
Linked Gene ◽  
Pcr Method

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

2006 ◽  
Vol 65 (3) ◽  
pp. 476-487 ◽  
Author(s):  
Miguel A. Providenti ◽  
Jason M. O'Brien ◽  
Robyn J. Ewing ◽  
E. Suzanne Paterson ◽  
Myron L. Smith
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Sybr Green ◽  
Quantitative Real Time Pcr ◽  
Genetic Elements

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

scholarly journals A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽  
2020 ◽  
Vol 10 (12) ◽  
Author(s):  
Siti Suriawati Badai ◽  
Omar Abd Rasid ◽  
Ghulam Kadir Ahmad Parveez ◽  
Mat Yunus Abdul Masani
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Oil Palm ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr
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