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 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.

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

2007 ◽  
Vol 70 (2) ◽  
pp. 341-347 ◽  
Author(s):  
A. GONZÁLEZ ◽  
S. BOTELLA ◽  
R. M. MONTES ◽  
Y. MORENO ◽  
M. A. FERRÚS
Keyword(s):  
Multiplex Pcr ◽  
Water Samples ◽  
Direct Detection ◽  
Pcr Amplification ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Culture Methods ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Wastewater Samples

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30°C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37°C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

scholarly journals Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

Simultaneous detection of Staphylococcus aureus enterotoxin C-producing strains from clinical and environmental samples by multiplex PCR assay

2010 ◽  
Vol 61 (3) ◽  
pp. 585-590 ◽  
Author(s):  
Geevaretnam Jeyasekaran ◽  
Kannan Thirumalai Raj ◽  
Robinsondhas Jeya Shakila ◽  
Albin Jemila Thangarani ◽  
Selvaramesh Karthika ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Multiplex Pcr ◽  
Environmental Samples ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

scholarly journals Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

2013 ◽  
Vol 79 (21) ◽  
pp. 6585-6592 ◽  
Author(s):  
Takayuki Miura ◽  
Sylvain Parnaudeau ◽  
Marco Grodzki ◽  
Satoshi Okabe ◽  
Robert L. Atmar ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Sewage Sample ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Reverse Transcription Pcr ◽  
Specificity And Sensitivity ◽  
Samples Selection

ABSTRACTNorovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

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 Development of a Multiplex PCR Assay for Detection and Discrimination of Pathogenic and Saprophytic Leptospira in Water

2021 ◽  
Author(s):  
Archana Vishwakarma ◽  
Gayathri Rethinavelu ◽  
Rathinsabapthi Pasupathi ◽  
Mohandass Ramya
Keyword(s):  
Multiplex Pcr ◽  
Water Samples ◽  
Tamil Nadu ◽  
Environmental Water ◽  
Causative Organism ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Pathogenic Leptospira ◽  
Multiplex Pcr Assay ◽  
Template Dna

Leptospirosis is a zoonosis prevalent in tropical countries and affects animals and humans alike. Leptospira interrogans, the causative organism for this waterborne infection, spreads through the urine of infected animals. There is a direct link between contaminated water and Leptospira outbreaks. This study reports a rapid assay to detect and differentiate pathogenic Leptospira from non-pathogenic in environmental water using multiplex PCR. The assay uses primers targeting the Lipl32 and Lipl21 gene. The multiplex PCR has been standardized using 11 pathogenic and one saprophytic serovar of Leptospira. The analytical sensitivity of the developed method was evaluated with different concentrations of template DNA. This method was used to screen water samples collected from 20 different sources from Chengalpattu town in Kancheepuram District, Tamil Nadu, India. Of the 20 water samples screened, 13 samples tested positive for pathogenic Leptospira, and seven samples tested negative. Four water samples were found to carry both pathogenic and saprophytic species. The developed multiplex PCR assay is highly useful for detecting and distinguishing pathogenic and saprophytic leptospires in water.

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.

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