Optimization of the performance of the polymerase chain reaction in silicon-based microstructures

134

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.

Section: Discussionmentioning

confidence: 99%

Application of the 5′-Nuclease PCR Assay in Evaluation and Development of Methods for Quantitative Detection of Campylobacter jejuni

Nogva¹,

Bergh²,

Holck³

et al. 2000

134

“…Most bacterial pathogens, including E. coli O157:H7, are present in very low numbers in soil, wastewater, and feces. Soil and feces also contain substances that are inhibitory to PCR and amplification efficiencies (31,32). The amplification efficiency and the efficiency of the probe cleavages have direct effects on the C T measurements of the DNA starting quantities (11,16,24).…”

Section: Specificity and Sensitivity Of Multiplex Real-time Pcr Assaymentioning

confidence: 99%

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification ofEscherichia coliO157:H7 in Dairy Wastewater Wetlands

Ibekwe

Watt

Grieve

et al. 2002

223

158

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C T ) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 ؋ 10 ؊5 pg of E. coli O157:H7 DNA ml ؊1 equivalent to approximately 6.4 ؋ 10 3 CFU of E. coli O157:H7 ml ؊1 based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >3.5 ؋ 10 4 CFU g ؊1 . E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g ؊1 with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.Constructed wetlands are commonly used for biotreatment of urban runoff and agricultural waste, such as dairy waste wash water from milk cows and on-farm waste from manure piles. These wastes harbor different bacterial species including human pathogens. Present management practices for dairy wash water on most commercial farms involve long-term storage in ponds where evaporation, percolation into groundwater, or spraying onto crops and/or disposal on land occurs. A constructed wetland treatment system maximizes the utility of existing storage ponds, reduces sediment loading in the wash water ponds through on-site treatment, increases pond capacity, and decreases the need to clean and scrape storage ponds. The goal is to have a final effluent water from the wetland that is suitable for on-site reuse and reduces the amount of contaminants entering groundwater supplies as a result of percolation of wash water stored in ponds and sprayed on disposal lands. This final product is expected to enhance water and air quality; reduce biological oxygen demand, nitrate-nitrogen, phosphorus, and ammonia emissions; and inactivate protozoan parasites and pathogens present in dairy wash water.The wash waters from dairy and beef cattle operations harbor different bacterial species including human pathogens such as enterohe...

“…All of these additional steps are time-consuming and laborious, and the additional procedures add to the overall cost of the test. The TaqMan assay utilizes the 5Ј-exonuclease activity of Thermus aquaticus DNA polymerase (17,21,35,43; K. J. Livak, L. Marmaro, and S. J. A.…”

mentioning

confidence: 99%

TaqMan PCR for Detection of Vibrio cholerae O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater

Lyon

2001

116

Vibrio cholerae is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for Vibrio is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of V. cholerae in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (hlyA) sequence of V. cholerae strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of V. cholerae tested and negative for all other species of Vibrio tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with V. cholerae serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g ؊1 and 10 CFU ml ؊1 in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the Vibrio cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of V. cholerae in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.Vibrio cholerae is a waterborne pathogen that causes gastrointestinal disorders with a wide range of clinical manifestations, including vomiting and rice-like diarrhea (24). The association of human illness with consumption of V. choleraecontaminated oysters, seawater, and other shellfish is well documented (29, 37). Consumption of raw oysters correlates strongly with gastrointestinal infections, and several Vibrio species, including strains of Vibrio parahaemolyticus, Vibrio vulnificus, and V. cholerae, have been implicated as the causative agents.Coastal areas with brackish waters and estuarine regions are niches for many Vibrio species, including strains of toxigenic O1 V. cholerae. Epidemic cholera strains are endemic in several regions, including the U.S. gulf coast and Australia, and are occasionally involved in illnesses in these regions (24). Because Vibrio species attach to material suspended in water, shellfish and mollusks that are in these environments can be expected to consume Vibrio during feeding (24).Traditional identification methods currently used are time-consuming and laborious, requiring prolonged incubation and selective enrichment to reduce the growth of background flora. Vibrio cells may also enter a viable but not culturable (VBNC) state, caused by nutrient starvation and physical stress. This may explain the failure of traditional culture techniques to isolate this organism from contaminated water and food samples implicated in food-borne outbreaks (10,27,47). Several investigator...