Comparison of mixed cell culture containing genetically engineered BGMK and CaCo-2 cells (Super E-Mix) with RT-PCR and conventional cell culture for the diagnosis of enterovirus meningitis

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of [Formula: see text]10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after [Formula: see text]10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required [Formula: see text]48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.Key words: RT-PCR, cell culture, ICC/PCR, enterovirus, hepatitis A virus.

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Rapid PCR-based monitoring of infectious enteroviruses in drinking water

Water Science & Technology ◽

10.2166/wst.1997.0771 ◽

1997 ◽

Vol 35 (11-12) ◽

pp. 423-427 ◽

Cited By ~ 8

Author(s):

K. S. Reynolds ◽

C. P. Gerba ◽

I. L. Pepper

Keyword(s):

Cell Culture ◽

Hepatitis A Virus ◽

Virus Detection ◽

Integrated Approach ◽

Rt Pcr ◽

Direct Pcr ◽

Integrated Method ◽

Cell Culture Assay ◽

Conventional Cell ◽

Reaction Volumes

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensitivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300–4001 of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24–48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48h against 5–16d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

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A convenient rapid culture assay for the detection of enteroviruses in clinical samples: comparison with conventional cell culture and RT-PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.47799-0 ◽

2008 ◽

Vol 57 (8) ◽

pp. 1000-1006 ◽

Cited By ~ 26

Author(s):

Elena Terletskaia-Ladwig ◽

Silvia Meier ◽

Ralph Hahn ◽

Michael Leinmüller ◽

Franz Schneider ◽

...

Keyword(s):

Cell Culture ◽

Clinical Samples ◽

Rt Pcr ◽

Conventional Cell

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Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

Water Science & Technology ◽

10.2166/wst.1995.0635 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 323-328 ◽

Cited By ~ 8

Author(s):

K. A. Reynolds ◽

C. P. Gerba ◽

I. L. Pepper

Keyword(s):

Cell Culture ◽

Polymerase Chain Reaction Amplification ◽

Cost Effective ◽

The United States ◽

Water Runoff ◽

Rt Pcr ◽

Marine Waters ◽

Storm Water Runoff ◽

Routine Monitoring ◽

Reaction Amplification

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

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Regulation of angiopoietin-like protein 4/fasting-induced adipose factor (Angptl4/FIAF) expression in mouse white adipose tissue and 3T3-L1 adipocytes

British Journal Of Nutrition ◽

10.1017/s0007114507882961 ◽

2008 ◽

Vol 100 (1) ◽

pp. 18-26 ◽

Cited By ~ 35

Author(s):

Sarah Dutton ◽

Paul Trayhurn

Keyword(s):

Skeletal Muscle ◽

Cell Culture ◽

Adipose Tissue ◽

White Adipose Tissue ◽

Culture Medium ◽

Mrna Level ◽

Mrna Levels ◽

Rt Pcr ◽

Before And After ◽

Stimulation Of

Angiopoietin-like protein 4 (Angptl4)/FIAF (fasting-induced adipose factor) was first identified as a target for PPAR and to be strongly induced in white adipose tissue (WAT) by fasting. Here we have examined the regulation of the expression and release of this adipokine in mouse WAT and in 3T3-L1 adipocytes. Angptl4/FIAF expression was measured by RT-PCR and real-time PCR; plasma Angptl4/FIAF and release of the protein in cell culture was determined by western blotting. The Angptl4/FIAF gene was expressed in each of the major WAT depots of mice, the mRNA level in WAT being similar to the liver and much higher (>50-fold) than skeletal muscle. Fasting mice (18 h) resulted in a substantial increase in Angptl4/FIAF mRNA in liver and muscle (9·5- and 21-fold, respectively); however, there was no effect of fasting on Angptl4/FIAF mRNA in WAT and the plasma level of Angptl4/FIAF was unchanged. The Angptl4/FIAF gene was expressed in 3T3-L1 adipocytes before and after differentiation, the level increasing post-differentiation; Angptl4/FIAF was released into the culture medium. Insulin, leptin, dexamethasone, noradrenaline, TNFα and several IL (IL-1β, IL-6, IL-10, IL-18) had little effect on Angptl4/FIAF mRNA levels in 3T3-L1 adipocytes. However, a major stimulation of Angptl4/FIAF expression was observed with rosiglitazone and the inflammatory prostaglandins PGD2 and PGJ2. Angptl4/FIAF does not act as an adipose tissue signal of nutritional status, but is markedly induced by fasting in liver and skeletal muscle.

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HLF is a Potential Prognostic Biomarker in Head and Neck Squamous Cell Carcinoma Based on Bioinformatic Analysis and Experimental Validation

10.21203/rs.3.rs-150871/v1 ◽

2021 ◽

Author(s):

Wei Fang ◽

Di Wan ◽

Jun Chen ◽

Weiqun Ma ◽

Zhen Luo ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Poor Prognosis ◽

Prognostic Biomarker ◽

Neck Squamous Cell Carcinoma ◽

Rt Pcr ◽

The Relationship

Abstract BackgroundHead and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers worldwide, with an increasing incidence. However, the underlying molecular mechanisms of HNSCC are poorly understood.MethodIn this work, 5 original datasets (GSE23558, GSE13601, GSE30784, GSE9844, GSE78060) of Head and neck squamous cell carcinoma (HNSCC) were selected from Gene Expression Omnibus (GEO) database. To identify differentially expressed genes (DEGs) in HNSCC and adjacent tissues. The common DEGs were acquired by Venn diagram. The sensitivity and specificity of HLF were determined by Receiver operating characteristic curves (ROC). Then, In order to further confirm the relationship between HLF and HNSCC patient’s prognosis, the expression and survival analysis of HLF was performed by Gene Expression Profiling Interactive Analysis (GEPIA), Cell culture, reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunohistochemical staining.ResultsSeventeen DEGs were screened from five sets of HNSCC functional gene expression series in GEO datasets. The low expression of HLF was indicated might be correlated with poor prognosis of HNSCC patients based on the bioinformatics analysis. According to the results of Cell culture, RT-PCR, western blotting, immunohistochemical staining, it was confirmed that the low level of HLF expression correlated with poor prognosis of HNSCC patients.ConclusionThe study effectively revealed useful information about the relationship of the low level of HLF expression and HNSCC. In summary, we identified HLF as a potential prognostic biomarker and therapeutic target for HNSCC.

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The Detection of Enteroviruses in Sewage Using Caco-2 Cells

Polish Journal of Microbiology ◽

10.33073/pjm-2013-014 ◽

2013 ◽

Vol 62 (1) ◽

pp. 97-100 ◽

Cited By ~ 3

Author(s):

MAGDALENA WIECZOREK ◽

ŁUKASZ KURYK ◽

AGNIESZKA WITEK ◽

ANNA DIUWE ◽

BOGUMIŁA LITWIŃSKA

Keyword(s):

Cell Culture ◽

Rt Pcr ◽

Pcr Assay ◽

Beef Extract

The work presented here demonstrates the utility of Caco-2 cells to detect enteroviruses in sewage. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by cell culture assays and RT-PCR. Enteroviruses were detected in all sewage samples, but only one sample was positive solely in RT-PCR assay. We proved that Caco-2 cells were more effective than RD and L20B cells in enterovirus isolation, depending on procedures used in the inoculation process.

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Detection of human enteric viruses in stream water with RT-PCR and cell culture

Journal of Water and Health ◽

10.2166/wh.2004.0004 ◽

2004 ◽

Vol 2 (1) ◽

pp. 37-47 ◽

Cited By ~ 10

Author(s):

Kimberly Denis-Mize ◽

G. Shay Fout ◽

Daniel R. Dahling ◽

Donna S. Francy

Keyword(s):

Cell Culture ◽

Membrane Filtration ◽

Stream Water ◽

False Negative ◽

Molecular Data ◽

Sampling Procedure ◽

Rt Pcr ◽

Quality Controls ◽

Pcr Method ◽

Virus Recovery

A multiplex RT-PCR method was used to measure virus occurrence at five stream water sites that span a range of hydroclimatic, water-quality, and land-use characteristics. The performance of the molecular method was evaluated in comparison with traditional cell culture and Escherichia coli membrane filtration assays. The study incorporated multiple quality controls and included a control for virus recovery during the sampling procedure as well as controls to detect potentially false-negative and false-positive data. Poliovirus recovery ranged from 16 to 65% and was variable, even in samples collected within the same stream. All five sites were positive for viruses by both molecular and cell culture-based virus assays. Enteroviruses, reoviruses, rotaviruses, and hepatitis A viruses were detected, but the use of the quality controls proved critical for interpretation of the molecular data. All sites showed evidence of faecal contamination, and culturable viruses were detected in four samples that would have met the US Environmental Protection Agency's recommended E. coli guideline for safe recreational water.

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St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652005000500008 ◽

2005 ◽

Vol 47 (5) ◽

pp. 281-285 ◽

Cited By ~ 48

Author(s):

Iray M. Rocco ◽

Cecília L.S. Santos ◽

Ivani Bisordi ◽

Selma M.C.N. Petrella ◽

Luiz E. Pereira ◽

...

Keyword(s):

Cell Culture ◽

Human Case ◽

Sao Paulo ◽

Encephalitis Virus ◽

Cell Culture Supernatant ◽

São Paulo ◽

Rt Pcr ◽

Igg Antibody ◽

Paulo State ◽

São Paulo State

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

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