scholarly journals Organic Substances Interfere with Reverse Transcription-Quantitative PCR-Based Virus Detection in Water Samples

2014 ◽  
Vol 81 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Akihiko Hata ◽  
Hiroyuki Katayama ◽  
Hiroaki Furumai
Keyword(s):  
Humic Acid ◽  
River Water ◽  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Water Samples ◽  
Virus Detection ◽  
Virus Concentration ◽  
Organic Substances ◽  
Rt Pcr ◽  
River Water Samples

ABSTRACTReverse transcription (RT)-PCR-based virus detection from water samples is occasionally hampered by organic substances that are coconcentrated during virus concentration procedures. To characterize these organic substances, samples containing commercially available humic acid, which is known to inhibit RT-PCR, and river water samples were subjected to adsorption-elution-based virus concentration using an electronegative membrane. In this study, the samples before, during, and after the concentration were analyzed in terms of organic properties and virus detection efficiencies. Two out of the three humic acid solutions resulted in RT-quantitative PCR (qPCR) inhibition that caused >3-log10-unit underestimation of spiked poliovirus. Over 60% of the organics contained in the two solutions were recovered in the concentrate, while over 60% of the organics in the uninhibited solution were lost during the concentration process. River water concentrates also caused inhibition of RT-qPCR. Organic concentrations in the river water samples increased by 2.3 to 3.9 times after the virus concentration procedure. The inhibitory samples contained organic fractions in the 10- to 100-kDa size range, which are suspected to be RT-PCR inhibitors. According to excitation-emission matrices, humic acid-like and protein-like fractions were also recovered from river water concentrates, but these fractions did not seem to affect virus detection. Our findings reveal that detailed organic analyses are effective in characterizing inhibitory substances.

scholarly journals Detection and Genetic Analysis of Human Sapoviruses in River Water in Japan

2010 ◽  
Vol 76 (8) ◽  
pp. 2461-2467 ◽  
Author(s):  
Masaaki Kitajima ◽  
Tomoichiro Oka ◽  
Eiji Haramoto ◽  
Hiroyuki Katayama ◽  
Naokazu Takeda ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Real Time ◽  
River Water ◽  
Water Samples ◽  
Detection Efficiency ◽  
Water Environment ◽  
Filter Method ◽  
Rt Pcr ◽  
Pcr Assay ◽  
River Water Samples

ABSTRACT We investigated the prevalence of sapoviruses (SaVs) in the Tamagawa River in Japan from April 2003 to March 2004 and performed genetic analysis of the SaV genes identified in river water. A total of 60 river water samples were collected from five sites along the river, and 500 ml was concentrated using the cation-coated filter method. By use of a real-time reverse transcription (RT)-PCR assay, 12 (20%) of the 60 samples were positive for SaV. SaV sequences were obtained from 15 (25%) samples, and a total of 30 SaV strains were identified using six RT-PCR assays followed by cloning and sequence analysis. A newly developed nested RT-PCR assay utilizing a broadly reactive forward primer showed the highest detection efficiency and amplified more diverse SaV genomes in the samples. SaV sequences were frequently detected from November to March, whereas none were obtained in April, July, September, or October. No SaV sequences were detected in the upstream portion of the river, whereas the midstream portion showed high positive rates. Based on phylogenetic analysis, SaV strains identified in the river water samples were classified into nine genotypes, namely, GI/1, GI/2, GI/3, GI/5, GI/untyped, GII/1, GII/2, GII/3, and GV/1. To our knowledge, this is the first study describing seasonal and spatial distributions and genetic diversity of SaVs in river water. A combination of real-time RT-PCR assay and newly developed nested RT-PCR assay is useful for identifying and characterizing SaV strains in a water environment.

Detection of adenoviruses and enteroviruses in tap water and river water by reverse transcription multiplex PCR

2000 ◽  
Vol 46 (5) ◽  
pp. 417-424 ◽  
Author(s):  
Hong Baek Cho ◽  
Seung-Hoon Lee ◽  
Jang-Cheon Cho ◽  
Sang-Jong Kim
Keyword(s):  
Multiplex Pcr ◽  
River Water ◽  
Reverse Transcription ◽  
Water Samples ◽  
Environmental Samples ◽  
Tap Water ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Environmental Application ◽  
Multiplex Pcr Assay

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to simultaneously detect adenoviruses and enteroviruses, both of which have attracted much attention as molecular indices of viral pollution in environmental samples. The method involves a reverse transcription step, followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and adenoviruses. The sensitivity of this assay was found to be similar to that of each monoplex PCR or RT-PCR assay, and to be consistent regardless of relative concentrations of adenoviruses and enteroviruses. To assess suitability and environmental application of the RT multiplex PCR assay, a total of 12 river water samples and 4 tap water samples were analyzed by RT multiplex PCR, each monoplex PCR or RT-PCR, and cell culture assay on the Buffalo Green Monkey kidney cell line. The sensitivity of the RT multiplex PCR was also found to be similar to that of each monoplex PCR in environmental samples. This suggests the RT multiplex PCR assay could be applied to the routine monitoring of viral pollution in environ mental waters.Key words: adenoviruses, enteroviruses, multiplex PCR, tap water.

scholarly journals Development of a Reverse Transcription-Quantitative PCR Assay for Detection of Salivirus/Klassevirus

2013 ◽  
Vol 79 (11) ◽  
pp. 3529-3532 ◽  
Author(s):  
Eiji Haramoto ◽  
Masaaki Kitajima ◽  
Mikie Otagiri
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Water Samples ◽  
Maximum Concentration ◽  
Pcr Assay ◽  
River Water Samples ◽  
Treated Sewage ◽  
Reverse Transcription Quantitative Pcr ◽  
Highly Sensitive ◽  
Quantitative Pcr Assay

ABSTRACTA broadly reactive and highly sensitive reverse transcription-quantitative PCR assay to detect salivirus/klassevirus was developed. By means of the developed assay, salivirus/klassevirus was detected in 13 (93%) raw sewage, 4 (29%) secondary-treated sewage, and 9 (16%) river water samples, with a maximum concentration of 9.7 × 106copies/liter.

Preliminary development and validation of a real-time reverse transcription PCR assay for the semi-quantification of viable Campylobacter jejuni in water samples

2009 ◽  
Vol 60 (12) ◽  
pp. 3151-3158 ◽  
Author(s):  
S. Lin ◽  
B. Gilpin ◽  
P. Scholes ◽  
E. Podivinsky ◽  
J. Klena ◽  
...  
Keyword(s):  
Real Time ◽  
River Water ◽  
Campylobacter Jejuni ◽  
Reverse Transcription ◽  
Water Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Probable Number ◽  
River Water Samples ◽  
Reverse Transcription Pcr

A TaqMan-based real-time reverse transcription PCR (RT-PCR) assay was developed for semi-quantification of viable Campylobacter jejuni in water samples. This preliminary assay is based on measuring the heat-shock induction of groEL messenger RNA (mRNA); the logic being that only viable cells can synthesise new mRNA. A 128-bp C. jejuni groEL DNA fragment was cloned and used as a positive control standard in TaqMan runs. The assay could detect as few as 13 genome equivalent copies of groEL plasmid DNA and 1–2 colony forming unit (CFU) of viable C. jejuni. A multi-step concentration technique was developed for collecting C. jejuni from large volumes of water samples. An average recovery of 20% (range: 8–47%) was obtained at spiking levels of 750–6,500 CFU per 10-litre of river water. Starting from concentration, the enumeration of viable C. jejuni in river water samples was achieved in approximately 12 hours. Quantification of viable C. jejuni in natural river water samples by this method showed similar trends to a culture-based double enrichment most probable number (MPN) -PCR method. There is potential to apply this fast, specific and sensitive semi-quantitative method to monitor a range of water samples for viable C. jejuni.

scholarly journals Genetic analysis of hepatitis A virus strains recovered from the environment and from patients with acute hepatitis

2001 ◽  
Vol 82 (12) ◽  
pp. 2955-2963 ◽  
Author(s):  
Sonia Pina ◽  
Maria Buti ◽  
Rosend Jardí ◽  
Pilar Clemente-Casares ◽  
Joan Jofre ◽  
...  
Keyword(s):  
Acute Hepatitis ◽  
River Water ◽  
Water Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Serum Samples ◽  
Genotype I ◽  
River Water Samples

The molecular epidemiology of hepatitis A virus (HAV) was studied by analysing HAV strains recovered from environmental water samples over a 7 year period and strains recovered from patients with acute hepatitis over a 5 year period. A total of 54 samples of raw domestic sewage and 66 samples of river water were collected. HAV particles were concentrated and detected by nested RT–PCR. HAV infection in patients with acute hepatitis was serologically diagnosed in 26 of 74 serum samples, which were also analysed by nested RT–PCR. HAV RNA was detected in 57·4% of sewage samples, 39·2% of Llobregat river water samples, 20% of Ter river water samples and 61·6% of serum samples. The HAV genomes detected were characterized further by directly sequencing a region of the 5′ non-translated region, the VP1/2A junction region and, in some samples, the 2B region. Results showed a 95% prevalence of genotype I, with nearly 50% being either subgenotype IA or subgenotype IB. Various strains were found simultaneously in both environmental and clinical samples. These strains were closely related to those described in distant geographical areas. Genotype IIIA was also found in 5% of sewage samples and in 12·5% of serum samples. Strains belonging to a common endemic genotype were not identified. The abundance of HAV in the environment produces a situation of sanitary risk, especially considering the low prevalence of antibodies in the young population.

Fabrication of dendritic nanoporous gold via a two-step amperometric approach: Application for electrochemical detection of methyl parathion in river water samples

Talanta ◽  
2021 ◽  
Vol 226 ◽  
pp. 122130
Author(s):  
Gilberto J. Silva Junior ◽  
Jéssica Soares Guimarães Selva ◽  
Anandhakumar Sukeri ◽  
Josué M. Gonçalves ◽  
Matias Regiart ◽  
...  
Keyword(s):  
River Water ◽  
Electrochemical Detection ◽  
Water Samples ◽  
Methyl Parathion ◽  
Nanoporous Gold ◽  
River Water Samples

Modified paramagnetic beads in a microfluidic system for the determination of ethinylestradiol (EE2) in river water samples

2010 ◽  
Vol 25 (6) ◽  
pp. 1376-1381 ◽  
Author(s):  
Noelia A. Martínez ◽  
Rudolf J. Schneider ◽  
Germán A. Messina ◽  
Julio Raba
Keyword(s):  
River Water ◽  
Water Samples ◽  
Microfluidic System ◽  
River Water Samples ◽  
Paramagnetic Beads
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