scholarly journals Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

2014 ◽  
Vol 80 (24) ◽  
pp. 7505-7511 ◽  
Author(s):  
Satoshi Ishii ◽  
Gaku Kitamura ◽  
Takahiro Segawa ◽  
Ayano Kobayashi ◽  
Takayuki Miura ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Water Samples ◽  
Treatment Plant ◽  
Environmental Water ◽  
Quantitative Information ◽  
Environmental Water Samples ◽  
Aichi Virus ◽  
Murine Norovirus ◽  
Water Safety ◽  
Viral Pathogens

ABSTRACTTo secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/μl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.

scholarly journals Development of a Quantitative PCR Assay for Arcobacter spp. and its Application to Environmental Water Samples

2018 ◽  
Vol 33 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Rajani Ghaju Shrestha ◽  
Yasuhiro Tanaka ◽  
Bikash Malla ◽  
Sarmila Tandukar ◽  
Dinesh Bhandari ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Water Samples ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Pcr Assay ◽  
Quantitative Pcr Assay

scholarly journals A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

2015 ◽  
Vol 81 (9) ◽  
pp. 3077-3085 ◽  
Author(s):  
Rupert Bliem ◽  
Sonja Schauer ◽  
Helga Plicka ◽  
Adelheid Obwaller ◽  
Regina Sommer ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Vibrio Cholerae ◽  
Quantitative Pcr ◽  
Water Samples ◽  
Dynamic Range ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Aquatic Environments ◽  
Ecological Studies ◽  
Content Type

ABSTRACTVibrio choleraeis a severe human pathogen and a frequent member of aquatic ecosystems. Quantification ofV. choleraein environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenicV. choleraein environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-correctedV. choleraeabundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 102to 2.3 × 104cell equivalents liter−1, whereas GR-corrected abundances ranged from 4.7 × 103to 1.6 × 106cell equivalents liter−1. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenicV. choleraein various aquatic environments for ecological studies as well as for risk assessment programs.

scholarly journals Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

2011 ◽  
Vol 77 (13) ◽  
pp. 4336-4343 ◽  
Author(s):  
Akihiko Hata ◽  
Hiroyuki Katayama ◽  
Masaaki Kitajima ◽  
Chettiyappan Visvanathan ◽  
Chea Nol ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Water Samples ◽  
Rna Extraction ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Acid Extraction ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Viral Nucleic Acid

ABSTRACTInhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

Improving Mycobacterium spp. detection in water: a new methodological proposal

2004 ◽  
Vol 4 (2) ◽  
pp. 103-106
Author(s):  
R. Santos ◽  
S. Gonçalves ◽  
F. Macieira ◽  
F. Oliveira ◽  
R. Rodrigues ◽  
...  
Keyword(s):  
Water Samples ◽  
Recovery Rate ◽  
Opportunistic Infections ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Sample Treatment ◽  
Detection Time ◽  
Fast Detection ◽  
Enrichment Step ◽  
Mycobacterium Spp

In recent years, non-tuberculous mycobacteria (NTM), once considered merely environmental saprophytes, have emerged as a major cause of opportunistic infections. There is no evidence of human-to-human transmission but they have been found in several environmental water samples. It is, therefore, of the utmost importance to develop methods of rapidly and accurately detecting non-tuberculous mycobacteria in water samples. To obtain a maximum recovery rate and a reduction of Mycobacterium spp. detection time in water samples, different decontamination, enrichment procedures and antibiotics supplements were tested before the inoculation into the Bactec® system. The proposed method of sample treatment (decrease in the decontamination time, followed for a peptone pre-enrichment step and an aztreonam and cefepime supplement) before the inoculation into the Bactec® system proved to be a good option for reliable and fast detection of Mycobacterium spp. in water samples.

Sign in / Sign up

Export Citation Format

Share Document