Rapid PCR-based monitoring of infectious enteroviruses in drinking water

1997 ◽  
Vol 35 (11-12) ◽  
pp. 423-427 ◽  
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.

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

2001 ◽  
Vol 47 (2) ◽  
pp. 153-157 ◽  
Author(s):  
Kelly A Reynolds ◽  
Charles P Gerba ◽  
Morteza Abbaszadegan ◽  
Ian L Pepper
Keyword(s):  
Cell Culture ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Detection ◽  
Rt Pcr ◽  
Specific Detection ◽  
Polymerase Chain ◽  
Conventional Cell ◽  
Polymerase Chain Reaction Methodology

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.

Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

1995 ◽  
Vol 58 (12) ◽  
pp. 1357-1362 ◽  
Author(s):  
LEE-ANN JAYKUS ◽  
RICARDO DE LEON ◽  
MARK D. SOBSEY
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Ammonium Bromide ◽  
Rt Pcr ◽  
Initial Inoculum ◽  
Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

scholarly journals Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes

Diseases ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 84
Author(s):  
Sabine Bock ◽  
Bernd Hoffmann ◽  
Martin Beer ◽  
Kerstin Wernike
Keyword(s):  
Real Time ◽  
Viral Genome ◽  
Virus Detection ◽  
World Health ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reaction Volume ◽  
Reaction Volumes ◽  
Health Organization ◽  
The Individual

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

scholarly journals Influence of environmental factors on virus detection by RT-PCR and cell culture

2000 ◽  
Vol 88 (4) ◽  
pp. 633-640 ◽  
Author(s):  
G.D. Lewis ◽  
S.L. Molloy ◽  
G.E. Greening ◽  
J. Dawson
Keyword(s):  
Cell Culture ◽  
Environmental Factors ◽  
Virus Detection ◽  
Rt Pcr

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.

Applications of in-vitro cell culture for measuring infectivity and inactivation of Cryptosporidium parvum

2004 ◽  
Vol 4 (2) ◽  
pp. 87-92
Author(s):  
P.A. Rochelle
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Rt Pcr ◽  
In Vitro Cell Culture ◽  
Infectivity Assay ◽  
Public Health Officials ◽  
Practical Tool ◽  
Cell Culture Assay ◽  
Evaluating Methods

Cryptosporidium parvum presents a significant problem for the water industry and public health officials because of its prevalence in sources of drinking water and its resistance to chlorine-based disinfectants; there is an urgent need for alternative, more effective disinfection strategies. Therefore, developing and evaluating methods for assessing the infectivity and inactivation of C. parvum oocysts are of paramount importance. Infectivity assays based on in-vitro cell culture have been developed as alternatives to human and animal-based assays to overcome ethical, cost, and practicality issues. Data obtained over a two-year period with an HCT-8 cell culture/RT-PCR infectivity assay generated an ID50 of 99 oocysts (95% CI: 84-117) and demonstrated that the cell culture assay was equivalent to the standard CD-1 mouse model for measuring infectivity of C. parvum oocysts. Aggregate data generated over two years using the HCT-8 cell culture/RT-PCR assay to measure UV disinfection of C. parvum demonstrated that 2.4 mJ/cm2 and 4.9 mJ/cm2 were necessary to achieve 1-log10 and 2-log10 inactivation, respectively. This work demonstrated that an HCT-8 cell culture-based infectivity coupled with RT-PCR for detecting C. parvum infections is a practical tool that can provide valuable information about the efficacy of disinfectants and the infectivity of oocysts in environmental waters.

Evaluation of TV cell line viral susceptibility using conventional cell culture techniques

Open Medicine ◽  
2006 ◽  
Vol 1 (1) ◽  
pp. 12-22 ◽  
Author(s):  
Coralia Bleotu ◽  
Carmen Diaconu ◽  
Mihaela Chivu ◽  
Irina Alexiu ◽  
Simona Ruta ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Virus Detection ◽  
Virus Growth ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Viral Susceptibility ◽  
Golden Standard ◽  
Conventional Cell

AbstractDespite the fact that a lot of methods have been developed for rapid virus detection, classic cell culture is still “the golden standard”. The range of viruses that can be isolated and cultured in cell line systems is often limited by the susceptibility of cells to support viral replication. Since the primary cell culture, the best cellular system available to support replication of a large number of viruses, is very expensive and diffcult to obtain, cell lines, which are easier to manipulate, are commonly used for virus growth and isolation.In two previous papers we described the TV cell line initiated by our team from a laryngeal tumor, which harbors human papillomavirus (HPV) gene sequences. In this paper we analyze its capacity to support virus replication. Depending on the virus, different cytopathic effects were produced. Comparison of viral effect observed on this cell line with the effect obtained on other cell lines has been performed. This cell line might be used in the clinical virology laboratory for virus isolation.

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