CHEMICAL COMPONENTS OF POLYMERASE CHAIN REACTION IN 18S rRNA FOR DETECTION OF Cryptosporidium FROM RIVER WATER SAMPLES

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

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Survival of and lacZ expression in recombainant Pseudomonas strains introduced into riwer water microcosms

Canadian Journal of Microbiology ◽

10.1139/m95-062 ◽

1995 ◽

Vol 41 (6) ◽

pp. 461-469 ◽

Cited By ~ 5

Author(s):

Kamtin Leung ◽

Jack T. Trevors ◽

Hung Lee

Keyword(s):

Polymerase Chain Reaction ◽

River Water ◽

Genetic Marker ◽

Water Samples ◽

Bacterial Population ◽

Initial Density ◽

Gene Cassette ◽

Lacz Expression ◽

Chain Reaction ◽

Polymerase Chain

The lacZY gene cassette inserted into the chromosome of Pseudomonas aeruginosa UG2Lr and Pseudomonas aureofaciens Ps3732RNL11 was used as a genetic marker to study the fate of the Pseudomonas strains in river water microcosms. Expression of the lacZ marker in UG2Lr and Ps3732RNL11 was detected in microcosms containing as few as 12 and 14 colony-forming units (cfu)/mL of river water, respectively, by fluorimetric measurement of the β-galactosidase activity against 4-methylumbelliferyl-β-D-galactoside as the substrate. The persistence of and lacZ expression in the Pseudomonas strains were monitored in sterile and nonsterile river water in the presence and absence of added nutrients by dilution plating and fluorimetry, respectively. After incubation for 10 d at 10 °C, culturable populations of strain UG2Lr in sterile water samples, with and without nutrient added, decreased from an initial density of 1.5 × 104 to 1.7 × 103 and 4.6 × 103 cfu/mL, respectively. Despite similar numbers of UG2Lr cells in the two treatments, expression of the lacZ marker in the surviving cells of the nutrient-supplemented sample was 24 times higher than in the cells of the unamended sample. In nonsterile water samples, the density of UG2Lr declined to less than 6 cfu/mL in 30 d regardless of the nutrient conditions. A nutrient supplement increased the growth of the native bacterial population but did not enhance growth of and lacZ expression in the bacteria. Polymerase chain reaction analysis showed a decrease in amplification signal indicating a genuine decline in viable UG2Lr cell density in the water samples. Ps3732RNL11 cells established themselves in both sterile and nonsterile water samples. In nonsterile water samples, Ps3732RNL11 maintained a density of 20–40% of the culturable bacterial population. However, lacZ expression in both UG2Lr and Ps3732RNL11 cells was undetectable in nonsterile water samples after 30 d.Key words: survival, genetic marker, lacZ expression, polymerase chain reaction, recombinant Pseudomonas strains.

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Using a real-time quantitative polymerase chain reaction (PCR) method for reliable enumeration of Aeromonas hydrophila in water samples

African Journal of Microbiology Research ◽

10.5897/ajmr2012.2491 ◽

2013 ◽

Vol 7 (19) ◽

pp. 2119-2126

Author(s):

Trakhna Faten ◽

Maaroufi Abderrazak ◽

Gadonna Widehem Pascale

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Water Samples ◽

Aeromonas Hydrophila ◽

Quantitative Polymerase Chain Reaction ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

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Diversity ofLeptospiraspp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

Journal of Tropical Medicine ◽

10.1155/2017/3760674 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 8

Author(s):

Chai Fung Pui ◽

Lesley Maurice Bilung ◽

Kasing Apun ◽

Lela Su’ut

Keyword(s):

Polymerase Chain Reaction ◽

Water Samples ◽

Urban Areas ◽

Environmental Samples ◽

Soil Samples ◽

Chain Reaction ◽

Prevalence Studies ◽

Pcr Method ◽

Polymerase Chain ◽

Soil And Water

Various prevalence studies onLeptospirain animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophyticLeptospirain selected animals and environments. This study was therefore conducted to detectLeptospiraspp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. PathogenicLeptospirawas present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. IntermediateLeptospirawas present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. SaprophyticLeptospirawas present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76Leptospiraspp. were isolated. Based on DNA sequencing, the dominantLeptospiraspp. circulating in urban areas of Sarawak are pathogenicLeptospira noguchii, intermediateLeptospira wolffiiserovar Khorat, and saprophyticLeptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence ofLeptospiraspp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

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