Development and Application of Real-Time Quantitative PCR

Williams, P. Mickey; Giles, Todd; Tucker, Ayly L.; Winer, Jane; Heid, Chris

doi:10.1007/978-1-4612-4164-5_18

Cited by 5 publications

(7 citation statements)

References 16 publications

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“…Recently, the development of new fluorescent techniques has, however, led to novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acid sequences. These methods involve the detection of a specific PCR product in a homogeneous solution without the need to open the amplification tubes after PCR (4). The results can be read in real time as the PCR product accumulates or at the end of the thermal cycling protocol directly from the amplification wells.…”

Section: Introductionmentioning

confidence: 99%

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Nurmi

Ylikoski

Soukka

et al. 2000

Nucleic Acids Research

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A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10 7 initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

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Section: Introductionmentioning

confidence: 99%

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Nurmi

Ylikoski

Soukka

et al. 2000

Nucleic Acids Research

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“…Quantitative PCR has several advantages over the conventional end-point PCR: qPCR increases the specificity by including an internal hybridization probe, reduces crosscontamination by including UNG (dUTP N-glycosylase) and eliminating post-PCR processing, determines the starting concentration of target sequence in the sample, and is less sensitive to PCR inhibitors. 14,20,26 In this report, the development of a qPCR assay (IS900 TaqMan) specific for M. a. paratuberculosis and its application to the study of M. a. paratuberculosis in conjunction with the ESP culture system II are described.…”

mentioning

confidence: 99%

Development and Application of Quantitative Polymerase Chain Reaction Assay Based on the ABI 7700 System (TaqMan) for Detection and Quantification of Mycobacterium Avium Subsp. Paratuberculosis

et al. 2002

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Abstract. Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) zooepidemicus).Even at high cell numbers (10 5 CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10 5 -10 6 CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 10 5 -10 6 CFU/ml within 2 weeks for heavy shedders, 3-4 weeks for medium, and 6-8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY. The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1-2 days for medium and heavy shedders and Ͼ4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.

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“…We chose the 23S rRNA gene as our internal standard because 90-95% (nearly corresponding to total RNA) of the RNA in bacteria is ribosomal RNA. However, the limitation of using 23S rRNA is the relatively large difference in gene expression between the reference gene and the gene of interest (Lantz et al 1998;Williams et al 1998). Another potential limitation is the differential degradation of the target transcript during RNA purification or storage (as observed in this work).…”

Section: Potential Errors In the Gene Expression Analysesmentioning

confidence: 95%

“…The hlyA gene expression was measured relative to the level of the 23S rRNA expression. Factors affecting this ratio are critical for the accuracy of the analysis (Williams et al. 1998).…”

Section: Determination Of the Accuracy Of The Quantitative Gene Exprementioning

confidence: 99%

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Subtyping Listeria monocytogenes through the combined analyses of genotype and expression of the hlyA virulence determinant

Rudi¹,

Nogva²,

Naterstad³

et al. 2003

J Appl Microbiol

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Aims: A major challenge for Listeria monocytogenes diagnostics is that this bacterium is ubiquitous in the environment, and that only a small fraction of the lineages are potential human pathogens. The aim of this work was to obtain a better subtyping of L. monocytogenes through utilization of combined analyses of genotype and the expression of the virulence determinant hlyA. Methods and Results: We investigated the effect of growth temperature and medium on the hlyA expression. The gene expression levels were determined by real-time quantitative reverse transcription PCR. The expression pattern of hlyA was highly diverse among the different strains tested. The expression ranged from repression to a 1000-fold induction for growth at 42°C, as compared with 0°C. The expression patterns were compared with the corresponding genotypes. There were surprisingly low correlations between the expression patterns and the genotype clusterings.This is exemplified for the virulent type strain NTNC 7973 and non-virulent type strain DSMZ 20600. These strains are genetically nearly identical, while the hlyA gene expression patterns are very different. Conclusions: The hlyA gene expression was highly diverse even within genetically clustered subgroups of L. monocytogenes. Consequently, the gene expression patterns can be used to further differentiate the strains within these genetic subgroups. Significance and Impact of the Study: A major limitation in the control of L. monocytogenes is that the current tools for subtyping are not accurate enough in determining the potential virulent strains. The impact of this study is that we have developed a subtyping approach that actually targets a virulence property.

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Development and Application of Real-Time Quantitative PCR

Cited by 5 publications

References 16 publications

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

Development and Application of Quantitative Polymerase Chain Reaction Assay Based on the ABI 7700 System (TaqMan) for Detection and Quantification of Mycobacterium Avium Subsp. Paratuberculosis

Subtyping Listeria monocytogenes through the combined analyses of genotype and expression of the hlyA virulence determinant

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