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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.

Mygind T, Birkelund S, Birkebaek NH, Østergaard L, Jensen JS, Christiansen G - BMC Microbiol. (2002)

Bottom Line: Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.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.These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but this needs to be confirmed further.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Aarhus, Denmark. mygind@biobase.dk

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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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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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.

Mentions: When performing quantitative real-time PCR, it is assumed that the PCR efficiency of the standard samples is the same as that of the unknown samples. This is the basis for the calculations made. Therefore, it is important to determine the efficiency in both standards samples and unknown samples. PCR efficiency depends e.g. on inhibition in the sample, how well primers and probes are designed, and how well the PCR conditions are optimised. PCR efficiency was determined in the reference samples described above and in standard samples. The standard samples were a 10-fold dilution series of purified genomic C. pneumoniae DNA with known concentrations. Threshold cycles obtained with the real-time PCR for duplicates of the reference and standard samples were plotted against log to the concentration of the samples (log to the concentration: arbitrary numbers with four-fold difference for the reference samples, as concentrations were unknown) (fig. 1). Two straight lines were drawn and the slopes were determined by linear regression to be -3.848 (standard error = 0.259) for the reference samples and -3.144 (standard error = 0.065) for the standard samples. The two slopes were found to be significantly different (z = 2.64, P = 0.008). Efficiency is derived from the idealized function for the amount of PCR product formed: N = N0 × En, where N is number of amplified molecules, N0 is the initial number of molecules, n is the number of amplification cycles and E is the efficiency which is ideally 2. The standard curves are derived from the function described above: n = -(1/log E) * log N0 + (log N/log E). Therefore, the slope of the line equals -(1/ log E) and the efficiency can be calculated from the slope. From the slopes the efficiency of the real-time PCR in the reference samples was determined to be 1.82 and in the standard samples to be 2.08. The fact that the efficiency is a little larger than 2 is probably caused by the linear regression analysis which does not take into account that the efficiency can maximally be 2. Because of the difference in efficiency between reference and standard samples an inaccuracy in the determined concentration of the reference samples is present. As the efficiency in the reference samples is lower, the concentration determined on basis of the standard samples will be an underestimation. This is seen in table 2, as it would be highly unlikely to be able to determine the lowest concentration 0.05 copies/μl if it was not an underestimation caused by different efficiencies. The underestimation factor can be estimated for each cycle number. At cycle number 30 and with the described difference in efficiency the concentration would be underestimated 23-fold (see fig. 2). Differences in efficiency of PCR can be caused by several factors e.g. inhibitors and storage of the sample, but different batches of primers, probes and enzymes might also influence the efficiency. The DNA for the reference samples was released by Proteinase K treatment only with no subsequent purification; therefore inhibitors might have been present. Furthermore, as a larger volume of reference sample (6.8 μl) than standard sample (2 μl) was used, this could increase inhibition. On the other hand, the amplification efficiency appeared to be identical for all dilutions as indicated by the regression line, so inhibition from the sample preparation might not be the explanation; the reference samples had been stored in polypropylene tubes for more than a month before analysis, and as DNA binds to polypropylene [12], this could also affect the efficiency observed as this effect alters the DNA concentrations especially in the dilute samples [12].


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.

Mygind T, Birkelund S, Birkebaek NH, Østergaard L, Jensen JS, Christiansen G - BMC Microbiol. (2002)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117782&req=5

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.
Mentions: When performing quantitative real-time PCR, it is assumed that the PCR efficiency of the standard samples is the same as that of the unknown samples. This is the basis for the calculations made. Therefore, it is important to determine the efficiency in both standards samples and unknown samples. PCR efficiency depends e.g. on inhibition in the sample, how well primers and probes are designed, and how well the PCR conditions are optimised. PCR efficiency was determined in the reference samples described above and in standard samples. The standard samples were a 10-fold dilution series of purified genomic C. pneumoniae DNA with known concentrations. Threshold cycles obtained with the real-time PCR for duplicates of the reference and standard samples were plotted against log to the concentration of the samples (log to the concentration: arbitrary numbers with four-fold difference for the reference samples, as concentrations were unknown) (fig. 1). Two straight lines were drawn and the slopes were determined by linear regression to be -3.848 (standard error = 0.259) for the reference samples and -3.144 (standard error = 0.065) for the standard samples. The two slopes were found to be significantly different (z = 2.64, P = 0.008). Efficiency is derived from the idealized function for the amount of PCR product formed: N = N0 × En, where N is number of amplified molecules, N0 is the initial number of molecules, n is the number of amplification cycles and E is the efficiency which is ideally 2. The standard curves are derived from the function described above: n = -(1/log E) * log N0 + (log N/log E). Therefore, the slope of the line equals -(1/ log E) and the efficiency can be calculated from the slope. From the slopes the efficiency of the real-time PCR in the reference samples was determined to be 1.82 and in the standard samples to be 2.08. The fact that the efficiency is a little larger than 2 is probably caused by the linear regression analysis which does not take into account that the efficiency can maximally be 2. Because of the difference in efficiency between reference and standard samples an inaccuracy in the determined concentration of the reference samples is present. As the efficiency in the reference samples is lower, the concentration determined on basis of the standard samples will be an underestimation. This is seen in table 2, as it would be highly unlikely to be able to determine the lowest concentration 0.05 copies/μl if it was not an underestimation caused by different efficiencies. The underestimation factor can be estimated for each cycle number. At cycle number 30 and with the described difference in efficiency the concentration would be underestimated 23-fold (see fig. 2). Differences in efficiency of PCR can be caused by several factors e.g. inhibitors and storage of the sample, but different batches of primers, probes and enzymes might also influence the efficiency. The DNA for the reference samples was released by Proteinase K treatment only with no subsequent purification; therefore inhibitors might have been present. Furthermore, as a larger volume of reference sample (6.8 μl) than standard sample (2 μl) was used, this could increase inhibition. On the other hand, the amplification efficiency appeared to be identical for all dilutions as indicated by the regression line, so inhibition from the sample preparation might not be the explanation; the reference samples had been stored in polypropylene tubes for more than a month before analysis, and as DNA binds to polypropylene [12], this could also affect the efficiency observed as this effect alters the DNA concentrations especially in the dilute samples [12].

Bottom Line: Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.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.These results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but this needs to be confirmed further.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Aarhus, Denmark. mygind@biobase.dk

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.

Show MeSH
Related in: MedlinePlus