scholarly journals Rapid Detection of Enteroviruses in Small Volumes of Natural Waters by Real-Time Quantitative Reverse Transcriptase PCR

2005 ◽  
Vol 71 (8) ◽  
pp. 4523-4530 ◽  
Author(s):  
Jed A. Fuhrman ◽  
Xiaolin Liang ◽  
Rachel T. Noble
Keyword(s):  
Los Angeles ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Natural Waters ◽  
Cost Effective ◽  
Environmental Water ◽  
Viral Contamination ◽  
Reverse Transcriptase Pcr ◽  
Qrt Pcr ◽  
Field Samples

ABSTRACT Despite viral contamination of recreational waters, only bacterial, not viral, indicators are monitored routinely, due to a lack of rapid and cost-effective assays. We used negatively charged filters to capture enteroviruses from seawater and freshwater. Viral RNA was extracted using a commercial kit, and the viruses were quantified by real-time quantitative reverse transcriptase PCR (qRT-PCR). Poliovirus (6.6 to 330,000 virus particles/ml) was added to samples from watersheds in Los Angeles, California, and analysis showed that with 50-ml samples, a cellulose acetate/nitrate (HA) filter yielded final recovery of 51% (r 2 = 0.99) in fresh water and 23% (r 2 = 0.90) in seawater. However, for additions of low levels of virus (more likely to represent field samples; <104 enterovirus particles/ml), the recovery was lower and more variable, with HA being best in freshwater (17%, r 2 = 0.97) and the type GF/F glass filter having higher average recovery in seawater (GF/F, 17%; r 2 = 0.93; HA 12%, r 2 = 0.87). The optimized method was used with 1-liter field samples from two very different freshwater “creeks” that drain into Santa Monica Bay, California: Topanga Creek (TC), a relatively pristine mountain creek, and Ballona Creek (BC), a concrete-lined urban storm drain. One TC site out of 10 and 2 BC sites out of 7 tested significantly positive for enteroviruses, with higher enterovirus concentrations in BC than in TC (ca. 10 to 25 versus 1 equivalent enterovirus particle/ml). The presented filtration-qRT-PCR approach is fast (<8 h from sampling to results), sensitive, and cost efficient and is promising for monitoring viral contamination in environmental water samples.

Novel Approach for Detecting Prohibited Species-Specific Central Nervous System Tissue Contamination in Meat by One-Step Real-Time Reverse Transcriptase PCR

2009 ◽  
Vol 72 (5) ◽  
pp. 1063-1069 ◽  
Author(s):  
M. M. NAGARAJAN ◽  
D. LONGTIN ◽  
C. SIMARD
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Compliance Monitoring ◽  
Reverse Transcriptase Pcr ◽  
Qrt Pcr ◽  
Tissue Contamination ◽  
One Step ◽  
Species Specific

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 102 to 109 copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

scholarly journals A Quantitative Real-Time RT-PCR Assay for the Detection ofVenezuelan equine encephalitis virusUtilizing a Universal Alphavirus Control RNA

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Ariel Vina-Rodriguez ◽  
Martin Eiden ◽  
Markus Keller ◽  
Winfried Hinrichs ◽  
Martin H. Groschup
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Encephalitis Virus ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr ◽  
Equine Encephalitis Virus ◽  
Copy Numbers ◽  
Field Samples ◽  
Western Equine Encephalitis Virus ◽  
Western Equine Encephalitis

Venezuelan equine encephalitis virus(VEEV) is anAlphavirusfrom the familyTogaviridaethat causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome ofEastern equine encephalitis virus(EEEV),Western equine encephalitis virus(WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

scholarly journals HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

2022 ◽  
Vol 8 ◽  
Author(s):  
Ephraim Fass ◽  
Gal Zizelski Valenci ◽  
Mor Rubinstein ◽  
Paul J. Freidlin ◽  
Shira Rosencwaig ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Cost Effective ◽  
Developed Countries ◽  
Spike Protein ◽  
Medical Community ◽  
Effective Vaccine ◽  
Gene Variants ◽  
Spike Gene ◽  
Field Samples

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in &lt; 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Validation of a Real-Time Reverse Transcriptase–PCR Assay for the Detection of H7 Avian Influenza Virus

2010 ◽  
Vol 5 (s1) ◽  
pp. e148-e149
Author(s):  
Janice Pedersen ◽  
Mary Lea Killian ◽  
Nichole Hines ◽  
Dennis Senne ◽  
Brundaban Panigrahy ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Reverse Transcriptase ◽  
Avian Influenza Virus ◽  
Real Time ◽  
Pcr Assay ◽  
Reverse Transcriptase Pcr
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