Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae - PubMed (original) (raw)

Comparative Study

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

Tina Mygind et al. BMC Microbiol. 2002.

Abstract

Background: Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.

Results: We compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with chronic cough. We found the same detection limit for the two methods and clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA.

Conclusion: These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but this needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay work in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with chronic cough.

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Figures

Figure 1

Figure 1

Determination of the efficiency for the standard, reference and clinical samples. The threshold cycle is the cycle number at which the fluorescence curve for a given sample crosses the user-defined noise band. Log to the concentration for the reference samples is set as log to arbitrary numbers with four-fold difference as the reference samples are four-fold serial dilutions. The actual concentration is not needed when determining the efficiency as it depends on the slope of the line only.

Figure 2

Figure 2

Effect of difference in efficiency between standard samples and samples. This figure illustrates the error in determination of concentration by real-time PCR at cycle no. 30 when the efficiencies differ between standard samples and samples. Number of cycles: n, Efficiency standard samples: Estd, efficiency reference samples: Ers, Number of amplicons at cycle n: Nn, Number of amplicons at cycle zero: N0. When the two equations are divided by eachother it is seen that there is an 23-fold underestimation of the concentration when Estd = 2.0 and Ers = 1.8. From this calculation it is also evident that at higher cycle number the error in concentration determination is larger.

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