scholarly journals A new method for efficient detection of Cryptosporidium RNA by real-time reverse transcription-PCR with surfactants

2015 ◽  
Vol 15 (5) ◽  
pp. 1061-1068 ◽  
Author(s):  
Takahiro Sekikawa ◽  
Kosuke Toshiki
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
18S Rrna ◽  
Nonionic Surfactants ◽  
Sodium Dodecyl ◽  
Rrna Genes ◽  
Tween 20 ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Triton X

Cryptosporidium is one of the most common causes of waterborne diseases worldwide. Its oocysts possess a robust wall that is extremely resistant to the chlorine used for potable water disinfection. The current procedures of nucleic acid extraction and purification, such as the freeze–thaw (F/T) method and the commercial kits, are time consuming and expensive. To this end, a surfactant extraction treatment (SET) was developed as a method to extract nucleic acids from Cryptosporidium using only surfactants. The use of 18S rRNA improves the sensitivity of Cryptosporidium detection for real-time polymerase chain reaction (PCR), because 18S rRNA molecules are constitutively present in high copy numbers. Therefore, we applied SET to the detection of Cryptosporidium 18S rRNA using reverse transcription (RT)-PCR for the first time. RT-PCR was inhibited by 0.01% of the anionic surfactant sodium dodecyl sulfate (SDS), whereas the inhibition did not occur with 5% of the nonionic surfactants Tween 20, Triton X-100, Tween 80, and Triton X-114. However, the nonionic surfactants could not completely suppress the inhibition induced by 0.1% SDS. We successfully extracted 18S rRNA genes from oocysts by SET without the F/T method and detected them by real-time RT-PCR.

scholarly journals Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

2006 ◽  
Vol 72 (6) ◽  
pp. 3846-3855 ◽  
Author(s):  
M. Isabel Costafreda ◽  
Albert Bosch ◽  
Rosa M. Pint�
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Accurate Estimation ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Development Evaluation

ABSTRACT A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

Toxicity and biodegradability screening of nonionic surfactants using sediment-derived methanogenic consortia

1998 ◽  
Vol 38 (7) ◽  
pp. 55-62 ◽  
Author(s):  
Daniel H. Yeh ◽  
Kurt D. Pennell ◽  
Spyros G. Pavlostathis
Keyword(s):  
Methane Production ◽  
Organic Contaminants ◽  
Nonionic Surfactants ◽  
Sodium Dodecyl ◽  
Fatty Acid Esters ◽  
Tween 20 ◽  
Biological Compatibility ◽  
Chlorinated Organic ◽  
Tween 40 ◽  
Triton X

The objective of this study was to screen and select biologically-compatible surfactants for subsequent use in enhancing the bioavailability and reductive dechlorination of sorbed-phase chlorinated organic contaminants. Sixteen surfactants commonly used in surfactant-enhanced bioavailability experiments were examined: polyoxyethylene (POE) alcohols (Brij 30/35, Witconol SN-70/90/120), POE sorbitan fatty acid esters (Tween 20/21/40/60/61/65/80/81/85), the octylphenol ethoxylate Triton X-100, and the anionic sodium dodecyl sulfate. Two hexachlorobenzene-dechlorinating, mixed enrichment cultures -- one glucose-fed and the other lactate-fed -- were developed at 22°C using anaerobic media and contaminated estuarine sediment as inoculum. Serum bottle methanogenesis assays using these two cultures were performed at a surfactant concentration of 200 mg/L. Tween surfactants were found to be the least inhibitory and also partially degradable. Some Tween surfactants accelerated the rates of methanogenesis for the lactate-fed culture, but no enhancement was observed for the glucose-fed culture. Cultures amended with Tween 20, 21, and 80 had the slowest methane production, while those amended with Tween 40, 60, and 85 had the fastest methane production. All non-Tween surfactants inhibited methanogenesis. The results of this study indicate that the biological compatibility of nonionic surfactants, even those from the same homologous series, appear to be system-specific and can vary significantly.

scholarly journals Conductometric study of sodium dodecyl sulfate - nonionic surfactant (Triton X-100, Tween 20, Tween 60, Tween 80 or Tween 85) mixed micelles in aqueous solution

2012 ◽  
Vol 66 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Dejan Cirin ◽  
Mihalj Posa ◽  
Veljko Krstonosic ◽  
Maja Milanovic
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Synergistic Effect ◽  
Nonionic Surfactants ◽  
Mixed Micelles ◽  
Sodium Dodecyl ◽  
Tween 80 ◽  
Tween 20 ◽  
Dodecyl Sulfate ◽  
Tween 60 ◽  
Triton X

The present study is concerned with the determination of the critical micelle concentration (cmc) of mixed micelles of sodium dodecyl sulfate with one of five nonionic surfactants (Triton X-100, Tween 20, Tween 60, Tween 80 or Tween 85) from conductance measurements. Based on the calculated values of the ? parameters we have noticed that SDS-nonionic surfactants mostly showed strong synergistic effect. It was found that nonionic surfactants with mainly longer and more hydrophobic tail show stronger interactions with hydrophobic part of SDS, thus expressing stronger synergism. In SDS-Tween 80 binary system the strongest synergistic effect was noticed. SDS-Tween 85 micellar system showed antagonistic effect, most probably because the presence of the double bond in its three hydrophobic tails (three C18 tails) makes it sterically rigid.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1091
Author(s):  
Ali A. Rabaan ◽  
Raghavendra Tirupathi ◽  
Anupam A Sule ◽  
Jehad Aldali ◽  
Abbas Al Mutair ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Disease Severity ◽  
False Negative ◽  
Influential Factors ◽  
Confirmatory Test ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Viral Kinetics ◽  
Ct Value

Real-time RT-PCR is considered the gold standard confirmatory test for coronavirus disease 2019 (COVID-19). However, many scientists disagree, and it is essential to understand that several factors and variables can cause a false-negative test. In this context, cycle threshold (Ct) values are being utilized to diagnose or predict SARS-CoV-2 infection. This practice has a significant clinical utility as Ct values can be correlated with the viral load. In addition, Ct values have a strong correlation with multiple haematological and biochemical markers. However, it is essential to consider that Ct values might be affected by pre-analytic, analytic, and post-analytical variables such as collection technique, specimen type, sampling time, viral kinetics, transport and storage conditions, nucleic acid extraction, viral RNA load, primer designing, real-time PCR efficiency, and Ct value determination method. Therefore, understanding the interpretation of Ct values and other influential factors could play a crucial role in interpreting viral load and disease severity. In several clinical studies consisting of small or large sample sizes, several discrepancies exist regarding a significant positive correlation between the Ct value and disease severity in COVID-19. In this context, a revised review of the literature has been conducted to fill the knowledge gaps regarding the correlations between Ct values and severity/fatality rates of patients with COVID-19. Various databases such as PubMed, Science Direct, Medline, Scopus, and Google Scholar were searched up to April 2021 by using keywords including “RT-PCR or viral load”, “SARS-CoV-2 and RT-PCR”, “Ct value and viral load”, “Ct value or COVID-19”. Research articles were extracted and selected independently by the authors and included in the present review based on their relevance to the study. The current narrative review explores the correlation of Ct values with mortality, disease progression, severity, and infectivity. We also discuss the factors that can affect these values, such as collection technique, type of swab, sampling method, etc.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

scholarly journals Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing

2009 ◽  
Vol 21 (5) ◽  
pp. 679-683 ◽  
Author(s):  
Pamela J. Ferro ◽  
Jason Osterstock ◽  
Bo Norby ◽  
Geoffrey T. Fosgate ◽  
Blanca Lupiani
Keyword(s):  
Polymerase Chain Reaction ◽  
Avian Influenza ◽  
Real Time ◽  
High Throughput ◽  
Reverse Transcription ◽  
Virus Isolation ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Response Planning ◽  
Polymerase Chain

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384–well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96– and 384–well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96– and 384–well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96– and 384–well assays, respectively. The data presented herein supports the utility of the 384–well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

scholarly journals Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

2002 ◽  
Vol 68 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
Camile Pizeta Semighini ◽  
Mozart Marins ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Quantitative Analysis ◽  
Real Time ◽  
Aspergillus Nidulans ◽  
Abc Transporter ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Reverse Transcription Pcr ◽  
Nitroquinoline Oxide

ABSTRACT The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.

scholarly journals bdrF2 of Lyme Disease Spirochetes Is Coexpressed with a Series of Cytoplasmic Proteins and Is Produced Specifically during Early Infection

2005 ◽  
Vol 187 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Hongming Zhang ◽  
Abayami Raji ◽  
Michael Theisen ◽  
Paul R. Hansen ◽  
Richard T. Marconi
Keyword(s):  
Real Time ◽  
Dna Helicase ◽  
Early Infection ◽  
Rt Pcr ◽  
Inner Membrane ◽  
Phase Partitioning ◽  
Multiple Promoters ◽  
Cytoplasmic Proteins ◽  
Rapid Amplification ◽  
Triton X

ABSTRACT The Bdr proteins are polymorphic inner membrane proteins produced by most Borrelia species. In Borrelia burgdorferi B31MI, the18 bdr genes form three subfamilies, bdrD, bdrE, and bdrF. The production of at least one of the Bdr paralogs, BdrF2, is up-regulated in host-adapted spirochetes, suggesting a role for the protein in the mammalian environment. Here, we demonstrate using reverse transcriptase (RT) PCR that BBG29, BBG30, BBG31, and BBG32, which reside upstream of bdrF2 , are cotranscribed with bdrF2 as a five-gene operon. While the functions of most of these proteins are unknown, BBG32 encodes a putative DNA helicase. Real-time RT-PCR analyses demonstrated higher levels of bdrF2 transcript relative to other genes of the operon, suggesting that bdrF2 may also be transcribed independently from an internal promoter. Internal promoters were detected using the 5′ rapid amplification of cDNA ends system. The putative promoter associated with bdrF2 was found to be highly similar in sequence to the multiple promoters associated with the ospC gene. Real-time RT-PCR analyses, performed to assess the expression of these genes in infected mice, revealed that genes of the bdrF2 locus are expressed only during early infection, suggesting a role in the establishment of infection. To further characterize the proteins encoded by the bdrF2 locus, which have unknown functions, the cellular localizations of these proteins were determined by Triton X-114 extraction and phase partitioning. BBG29 and BBG31 were found to be cytoplasmic. To determine if these proteins elicit an antibody (Ab) response during infection, immunoblot analyses were performed. Abs to these proteins were not detected. Based on the analyses presented here, we offer the hypothesis that BdrF2 and other proteins encoded by the operon form an inner-membrane-associated protein complex that may interact with DNA and which carries out its functional role during transmission or the early stages of infection.

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