Nested Quantitative Real Time PCR for Detection of Occult Tumor Cells

Max, Nicole; Wolf, K.; Thiel, Eckhard; Keilholz, Ulrich

doi:10.1007/978-3-642-59537-0_3

Cited by 19 publications

(11 citation statements)

References 9 publications

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“…Results have been mixed, with some investigators finding that RT-PCR detection of tyrosinase in peripheral blood samples correlates with prognosis and others finding no correlation (Table 1). More recently, quantitative real-time polymerase chain reaction (QRT-PCR) has been used to determine the relative level of circulating melanoma cells [23,24]. A direct correlation between outcome and circulating tumor cell burden is still lacking in most studies, but some large multi-institutional trials are still in progress, and we need to await these results before coming to a conclusion on the usefulness of detecting circulating melanoma cells by the detection of tyrosinase using RT-PCR or QRT-PCR techniques.…”

Section: Tyrosinasementioning

confidence: 99%

Tumor cell and circulating markers in melanoma: Diagnosis, prognosis, and management

Kounalakis

Goydos

2005

Curr Oncol Rep

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The search is on for biomarkers for use in the diagnosis, staging, prognosis, and management of patients with melanoma. As with many types of cancer, the hematogenous spread of melanoma is a bad prognostic sign, and many groups have attempted to detect circulating melanoma cells in patients with different stages of melanoma. Some studies have used direct extraction of intact tumor cells from the peripheral blood and others the detection of surrogate markers of circulating melanoma cells, such as tyrosinase or MART-1. However, a correlation between the detection of intact melanoma cells in the circulation and prognosis is controversial. Many other biomarkers have also been studied, including lactate dehydrogenase, S100, TA90, and C-reactive protein. Much progress has been made, and preliminary studies have shown promise with many of these markers. Finally, the detection of tumor-specific circulating DNA has shown promise as a prognostic and diagnostic marker of disease in melanoma as well. In this review we examine the most promising biomarkers for use in patients with cutaneous melanoma.

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Section: Tyrosinasementioning

confidence: 99%

Tumor cell and circulating markers in melanoma: Diagnosis, prognosis, and management

Kounalakis

Goydos

2005

Curr Oncol Rep

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“…Nested quantitative PCR has been described previously (5,22), but it has not been widely adopted as an RTQ-PCR strategy. Inglis and Kalischuk (8) modified the primer set of Logan et al (19) based on the 16S rRNA gene and used nPCR to detect C. lanienae in cattle.…”

Section: Nested Nonnestedmentioning

confidence: 99%

Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR

Inglis

Kalischuk

2004

Appl Environ Microbiol

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Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies (Ϸ3 ؋ 10 3 CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (Ϸ250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.Campylobacter species are recognized as one the most frequent causes of acute diarrheal disease in humans in North America (Centers for Disease Control and Prevention-U.S. Department of Agriculture-Food and Drug Administration Collaborating Sites Foodborne Disease Active Survey Network [Foodnet; http://www.cd.gov/foodnet/annuals.htm]). Alberta, Canada, possesses a very large beef cattle population (Ϸ5 million head) centered in the Southern region of the province, but relatively limited research has investigated the prevalence of Campylobacter species associated with beef cattle. The prevalence of Campylobacter infections in humans in this region is considerably higher than the national average (Health Canada website [http://dsol-smed.hc-sc.gc.ca/dsol-smed/ndis/index_e. html]), and cattle in this region shed a variety of Campylobacter species (8). In a PCR-based survey of 380 feedlot steers, 83% of the animals were observed to be shedding campylobacters, and the most prevalent taxa were Campylo...

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“…To increase the sensitivity to a target DNA copy number of 1 or 10; a combination involving nested PCR is currently being developed. Recently, a nested PCR strategy has been used to increase the sensitivity of real-time quantitative PCR for detection of occult tumor cells [24].…”

Section: Competitive Internal Standard Dna and Pcr E¤ciencymentioning

confidence: 99%

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

Widada¹

2001

FEMS Microbiology Letters

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The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

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Nested Quantitative Real Time PCR for Detection of Occult Tumor Cells

Cited by 19 publications

References 9 publications

Tumor cell and circulating markers in melanoma: Diagnosis, prognosis, and management

Tumor cell and circulating markers in melanoma: Diagnosis, prognosis, and management

Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

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