scholarly journals Quantitative detection of enteroviruses in activated sludge by cell culture and real-time RT-PCR using paramagnetic capturing

2005 ◽  
Vol 3 (3) ◽  
pp. 313-324 ◽  
Author(s):  
D. Pusch ◽  
St. Ihle ◽  
I. Graeber ◽  
J. M. López-Pila ◽  
M. Lebuhn
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Real Time ◽  
Genomic Region ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Affinity Capture ◽  
Direct Capture ◽  
Cytopathic Effects ◽  
Green Monkey

We have compared in extracts of activated sludge the number of enteroviruses detectable with buffalo green monkey (BGM) cell-cultures versus the number of enteroviral genomes determined by reverse-transcription quantitative real-time PCR (RT-qPCR). In order to find conditions adequate for quantifying enteroviral RNA isolated from (waste)water we have investigated affinity capture of RNA with polystyrene beads (Dynabeads). The capture efficiency strongly depended on the genomic region chosen for the affinity binding. Capture of the RNA by its 3′-tail was most efficient (almost 100%); other regions within the genome yielded variable but lower results. Indirect capture (first hybridization of the RNA to the oligonucleotides, then attachment of the duplex molecules to the beads) was much more efficient than direct capture (attachment of the oligonucleotides to the beads first, then binding of the RNA), and resulted in RNA capture of maximally 60–80%. At least partly, this was due to incomplete hybridization of the RNA to the complementary oligonucleotides. No correlation was found between the number of cytopathic effects (CPE) determined by cell culture and the number of genomes quantified by RT-qPCR; RT-qPCR values were consistently much higher than the number of CPE. This points to overestimation of infectious enteroviruses by RT-qPCR and/or underestimation by the cell culture approach.

Rapid Detection Method for Hepatitis A Virus from Lettuce by a Combination of Filtration and Integrated Cell Culture–Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (10) ◽  
pp. 1756-1761 ◽  
Author(s):  
JI-YEON HYEON ◽  
JUNG-WHAN CHON ◽  
CHANKYU PARK ◽  
JUNG-BOK LEE ◽  
IN-SOO CHOI ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Cytopathic Effects ◽  
Reverse Transcription Pcr ◽  
Charged Membrane ◽  
Polyethylene Glycol Precipitation ◽  
Positively Charged

We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture–real-time reverse transcription PCR (ICC–real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC–real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC–real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC–real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC–real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC–real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

Rapid and quantitative detection of mumps virus RNA by one-step real-time RT-PCR

2004 ◽  
Vol 49 (2) ◽  
pp. 83-88 ◽  
Author(s):  
Ayhan Kubar ◽  
Mehmet Yapar ◽  
Bulent Besirbellioglu ◽  
I.Yasar Avci ◽  
Cakır Guney
Keyword(s):  
Real Time ◽  
Mumps Virus ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Virus Rna ◽  
One Step

Cell culture demonstrates superior sensitivity over one step real time RT PCR and nested VP1 amplification for Enteroviruses

2021 ◽  
Vol 287 ◽  
pp. 113994
Author(s):  
Auwal Idris Kabuga ◽  
Ahmad Nejati ◽  
Parastoo Soheili ◽  
Soodeh Yousefipoor ◽  
Maryam Yousefi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Rt Pcr ◽  
One Step

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

Quantitative Detection of AML1-ETO Rearrangement by Real-Time RT-PCR Using Fluorescently Labeled Probes

2001 ◽  
Vol 42 (4) ◽  
pp. 747-756 ◽  
Author(s):  
E. Barragán ◽  
P. Bolufer ◽  
I. Moreno ◽  
G. Martín ◽  
J. Nomdedeu ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Detection ◽  
Rt Pcr

scholarly journals Severe Acute Respiratory Syndrome Coronavirus-2 Inactivation Activity of the Polyphenol-Rich Tea Leaf Extract with Concentrated Theaflavins and Other Virucidal Catechins

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4803
Author(s):  
Yohei Takeda ◽  
Kyohei Tamura ◽  
Dulamjav Jamsransuren ◽  
Sachiko Matsuda ◽  
Haruko Ogawa
Keyword(s):  
Cell Culture ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Western Blotting ◽  
Leaf Extract ◽  
Culture Media ◽  
Control Measures ◽  
Virucidal Activity ◽  
Rt Pcr ◽  
Tea Leaf

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is producing a large number of infections and deaths globally, the development of supportive and auxiliary treatments is attracting increasing attention. Here, we evaluated SARS-CoV-2-inactivation activity of the polyphenol-rich tea leaf extract TY-1 containing concentrated theaflavins and other virucidal catechins. The TY-1 was mixed with SARS-CoV-2 solution, and its virucidal activity was evaluated. To evaluate the inhibition activity of TY-1 in SARS-CoV-2 infection, TY-1 was co-added with SARS-CoV-2 into cell culture media. After 1 h of incubation, the cell culture medium was replaced, and the cells were further incubated in the absence of TY-1. The viral titers were then evaluated. To evaluate the impacts of TY-1 on viral proteins and genome, TY-1-treated SARS-CoV-2 structural proteins and viral RNA were analyzed using western blotting and real-time RT-PCR, respectively. TY-1 showed time- and concentration-dependent virucidal activity. TY-1 inhibited SARS-CoV-2 infection of cells. The results of western blotting and real-time RT-PCR suggested that TY-1 induced structural change in the S2 subunit of the S protein and viral genome destruction, respectively. Our findings provided basic insights in vitro into the possible value of TY-1 as a virucidal agent, which could enhance the current SARS-CoV-2 control measures.

scholarly journals Quantitative detection of Citrus tristeza virus in plant tissues and single aphids by real-time RT-PCR

2007 ◽  
Vol 120 (2) ◽  
pp. 177-188 ◽  
Author(s):  
Edson Bertolini ◽  
Aranzazu Moreno ◽  
Nieves Capote ◽  
Antonio Olmos ◽  
Ana de Luis ◽  
...  
Keyword(s):  
Real Time ◽  
Citrus Tristeza Virus ◽  
Quantitative Detection ◽  
Plant Tissues ◽  
Rt Pcr ◽  
Tristeza Virus ◽  
Citrus Tristeza
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