< > Home : FAQ : Professional : Chlamydiales : Immunobiology : Infections : Diagnosis & Treatment : Links : Contact Us

Quantitative PCR offers significant advantages over conventional pcr in its rapidity, the ease with which it can be automated, the potential decreased risk of carry-over contamination and the provision of a quantitative result. The latter is only truly quantitative if PCR efficiency approaches 100%; i.e. there are no inhibitors present.

The table below summarises some of the main publications on quantitative pcr of chlamydiae. References to the recent Antalya proceedings have been included, on the grounds that these papers are peer reviewed and likely to appear in fuller form as conventional publications. Where possible the hyperlink from the lead author name in the table goes to the original publication or to the PubMed abstract.

Organism 1. Chlamydophila spp	Gene (s)	Methodology	Comment	Author / Reference / Link
C. pneumoniae	pmp4	LightCycler ®	Sensitivity approx 1 genome copy per ul. Used on paraffin embedded formalin fixed tissue. Correlated well with immunohistochemistry	Mygind et al., 2001
C. pneumoniae	16S rRNA	TaqMan  ®	Used an 185 bp amplicon & Sybr Green. Also describes RT-PCR on dnA, polA, mutS & minD genes.  Found that IFN-g - mediated persistence involved production of transcripts associated with DNA replication but not cell division.	Byrne et al., 2001
C. pneumoniae	rpoB	TaqMan  ®	100 bp amplicon and exonuclease probe. No positives from acute coronary syndrome patients, reasons not clear.	Kiss et al., 2001
C. pneumoniae	Cloned pst1 fragment	LightCycler ®	17% positive on PBMCs from coronary artery disease patients versus 1% from controls. 437 bp amplicon.	Leowattana et al., 2001
C. pneumoniae	pmp4	LightCycler ®	Method as above. The quantitative pcr and conventional pcr had similar performance on clinical samples	Mygind et al., 2002
C. pneumoniae	omp1	TaqMan	Diagnostic efficiency for respiratory tract infection similar or better to Tong & Sillis nested touchdown pcr, at 1- 5 copies. Acetyl-cysteine treated sputum samples provided highest copy numbers, perhaps because higher number of cells and larger volume of sample.	Kuoppa et al., 2002
C. pneumoniae	16S rRNA	TaqMan ®	Copy number was not correlated with severity of atheromatous plaque	Berger et al., 2000
C. pneumoniae	16S rRNA, omp1, pst1 cloned fragment	TaqMan ®	A careful comparison of omp1 qPCR with conventional nested / touchdown pcrs. qPCR performed at least as well, was time saving and less prone to contamination. Lack of concordance of pcrs overall on clinical specimens. Further studies of qPCR needed.	Apfalter et al., 2003
C. pneumoniae	16S rRNA, 16S - 23S rRNA spacer	LightCycler ®	Sensitivity similar to Tong & Sillis touchdown nested PCR at 0.5 IFU/pcr. DNA loss on freezing. Reagents costly.	Hardick et al., 2002
C. pneumoniae	13 membrane proteins	Rotorgene 2000	Preliminary report of comparison of expression of membrane protein genes during acute and continuous infection.	Hogan et al., 2002
C. pneumoniae	omp1	TaqMan  ®	Particularly comprehensive study. Tested two different primer sets for real time PCR. Compared with conventional PCR & tested all PCRs on clinical specimens. Real time PCR had excellent detection limits but on clinical methods there were discrepancies between all methods and number of positive samples was low. Amplicon sizes of 108 or 125 base pairs. Used external control amplification.	Tondella et al., 2002
C. pneumoniae		LightCycler ®	Assessed analytical sensitivity and specificity on respiratory specimes using criteria of Centers for Disease Control and Prevention (USA) and Laboratory Centre for Disease Control (Canada). Analytical sensitivity of 1 inclusion forming unit per ml of bronchoalveolar lavage fluid, or 0.02 inclusion forming units per PCR reaction, reported. 100% specificity and sensitivity testing DNA preparations from 12 specimens of patients with known pulmonary Chlamydia pneumoniae infection and from 78 specimens of patients with respiratory tract disease of other origin.	Reischl et al., 2003
C. pneumoniae	Cpn0278, ArgR		Compares respiratory and atherosclerosis associated strains. Includes qPCR for Mycoplasma contaminants. Detected 10-4 IFU. Used on respiratory samples.	Bonanomi et al., 2003
C. abortus	omp1, rRNA	LightCycler ®	Copy number correlated well with IFU	Huang et al., 2001
C. abortus, C. pecorum	23S rRNA, omp1, omp1-RT, 16S-23S rRNA intergen /RT	LightCycler ®	Detailed study of parameters. Freeze thawing damages chlamydial DNA. Collect / stabilise DNA in guanidium thiocyanate. High Pure extraction preferred to Wizard or QIAmp. 23SrRNA target optimal for high sensitivity detection of genomic rRNA, particularly RT-qPCR. Step-down cycling preferred. 53% prevalence of low-level C. abortus and C. pecorum genital infection in virgin heifers suggested extragenital transmission and conformed to presence of specific antibody.	deGraves et al., 2003a, b
C. felis	omp1	iCycler ®	Closed tube system decreases contamination. Able to detect fewer than 10 genomic copies.	Helps et al., 2001
2. Chlamydia spp
C. trachomatis	Sigma factors: rpoD, rpoN, rpsD	LightCycler ®	The sigma factors exhibited different patterns of temporal expression.	Mathews et al., 1999
C. trachomatis	8% of ORFs of genome		Used quantitative competitive PCR and RT-PCR. Interesting approach to define developmental cycle-related transcripts	Shaw et al., 2000
C. trachomatis	rRNA	TaqMan	Innovative quantitative peptide nucleic acid (PNA) probe method	Fiandaca et al., 2001
C. trachomatis	omp1A	LightCycler ®	Several papers in Antalya proceedings describing qPCR in trachoma field studies. Primers not defined. Association of high load with active infection, particularly TI. Many active cases with low load. See Solomon et al., 2003.	Solomon et al., 2002
C. trachomatis	omp1	ABI Genscan 672	Early attempt to quantitate chlamydiae using Genescan software.	Pecharatana et al., 1993
C. trachomatis	omp1	LightCycler ®	Prescreen with Amplicor PCR. Field study of trachoma. Constant domain 3 of the omp1 gene. Primer sequences: CT1: 5'  GCTGTGGTTGAGCTTTATACAGACAC-3'; CT2: 5'  TTTAGGTTTAGATTGAGCATATTGGA-3'	Solomon et al., 2003
Multiplex for C. pneumoniae, M. pneumoniae and L. pneumophila	various	TaqMan	Performed in two separate reactions. Comparison with conventional PCR on 73 respiratory specimens showed agreement of  95.8%, 100% and 100% for C. pneumoniae, L. pneumophila and M. pneumoniae. Of 19 serology-positive patients, 14 were confirmed by the multiplex PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative by multiplex PCR.	Welti et al., 2003

An incoming commercial form of real time PCR is the Abbott molecular beacons test system.

A database for primers and probes for real time PCR is available on the web [Pattyn et al., 2003] [but suffers from irritating pop up advertising]. The database may be queried using the official gene name, a sequence, Locus link or SNP identifier. Researchers are encouraged to submit their validated primer and probe sequences.

[MEW] June 2003

[I am grateful to Tina Mygind (Aarhus) who made a number of very valuable suggestions on this article which have been incorporated].

SEE ALSO: Diagnostics: Molecular Beacons

NEXT: PCR speciation

Index

References

Apfalter, P., Barousch, W., Nehr, M., Makristathis, A., Willinger, B., Rotter, M. & Hirschl, A. M. (2003). Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays. Journal of Clinical Microbiology 41, 592 - 600.

Berger, M., Schroder, B., Daeschlein, G., Schneider, W., Busjahn, A., Buchwalow, I., Luft, F. C. & Haller, H. (2000). Chlamydia pneumoniae DNA in non-coronary atherosclerotic plaques and circulating leukocytes. Journal of Laboratory and Clinical Medicine 136, 194 - 200.

Bonanomi, A., Dohm, C., Rickenbach, Z., Altwegg, M., Fischer, J., Gygi, D. & Nadal, D. (2003). Monitoring intracellular replication of Chlamydophila (Chlamydia) pneumoniae in cell cultures andcomparing clinical samples by real-time PCR. Diagnostic Microbiology and Infectious Disease 46, 39-47.

Byrne, G. I., Ouellette, S. P., Wang, Z., Rao, J. P., Lu, L., Beatty, W. L. & Hudson, A. P. (2001). Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells. Infection and Immunity 69,  5423 - 5429. Full article   

DeGraves, F. J., Gao, D. & Kaltenboeck, B. (2003a). High-sensitivity quantitative PCR platform. Biotechniques. 34, 106 - 110;  112 - 115.

DeGraves, F. J., Gao, D., Hehnen, H. R., Schlapp, T. & Kaltenboeck, B. (2003b). Quantitative Detection of Chlamydia psittaci and C. pecorum by High-Sensitivity Real-Time PCR Reveals High Prevalence of Vaginal Infection in Cattle. Journal of Clinical Microbiology 41, 1726 - 1729.

Fiandaca, M. J., Hyldig-Nielsen, J. J., Gildea, B. D. & Coull, J. M. (2001). Self-reporting PNA/DNA primers for PCR analysis. Genome Research 11, 609 - 613. Full article  

Hardick, J., Maldeis, N., Dalesio, N., Theodore, M., Wood, B., Quinn, T. & Gaydos, C. (2002). Real time pcr for Chlamydia pneumoniae based on existing primer set CPN90 and CPN91 for the Roche LightCycler. In Schachter et al., (eds) Chlamydial infections. Proceedings of the tenth international symposium on human chlamydial infections, pp 433 - 436. Published by International Chlamydia Symposium, San Francisco, CA 94110. ISBN 0-9664383-1-0

Helps, C., Reeves, N., Tasker, S. & Harbour, D. (2001). Use of real-time quantitative PCR to detect Chlamydophila felis infection. Journal of Clinical Microbiology 39, 2675 - 2676. Full article  

Hogan, R., Mathews, S., Kutlin, A., Hammerschlag, M. & Timms, P. (2002). Differential expression of genes encoding membrane proteins between acute and continuous Chlamydia pneumoniae infections. In Schachter et al., (eds) Chlamydial infections. Proceedings of the tenth international symposium on human chlamydial infections, pp 615 - 618. Published by International Chlamydia Symposium, San Francisco, CA 94110. ISBN 0-9664383-1-0

Huang, J., DeGraves, F. J., Gao, D., Feng, P., Schlapp, T. & Kaltenboeck, B. (2001). Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler. Biotechniques 30, 150 - 157.

Kiss, K., Khanakah, G., Kundi, M., Glogar, H. D. & Stanek, G. (2001). Increase of chlamydial LPS antibodies in patients with acute coronary syndrome without detection of chlamydial DNA in atherectomy samples. Wien Klin Wochenschr, 113,  731 - 736.

Kuoppa, Y., Boman, J., Scott, L., Kumlin, U., Eriksson, I. & Allard, A. (2002). Quantitative Detection of Respiratory Chlamydia pneumoniae Infection by Real-Time PCR. Journal of Clinical Microbiology 40, 2273 - 2274.

Leowattana, W., Pokum, S., Mahanonda, N., Poungvarin, N. (2001). Rapid detection of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells of coronary artery disease patients by real-time fluorescence PCR. Journal of the Medical Association of Thailand 84 Suppl 3,  S658 - S668.

Mathews, S. A., Volp, K. M. & Timms, P. (1999). Development of a quantitative gene expression assay for Chlamydia trachomatis identified temporal expression of sigma factors. FEBS Letters 458, 354 - 358.

Mygind, T., Birkelund, S., Falk, E. & Christiansen, G. (2001). Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry. Journal of Microbiological Methods 46, 241 - 251.

Mygind, T., Birkelund, S., Birkebaek, N., Oestergaard, L., Jensen, J. & Christiansen, G. (2002). 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. Biomed Central Microbiology 2, 17 - 24. Full article  

Pattyn, F., Speleman, F., De Paepe, A. & Vandesompele, J. (2003). RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Research 31, 122 - 123. Web link to database

Pecharatana, S., Pickett, M. A., Watt, P. J. & Ward, M. E. (1993). Genotyping ocular strains of Chlamydia trachomatis by single-tube nested PCR. PCR Methods and Applications 3, 200 - 204.

Reischl, U., Lehn, N., Simnacher, U., Marre, R. & Essig, A. (2003). Rapid and standardized detection of Chlamydia pneumoniae using LightCycler real-time fluorescence PCR. European Journal of Clinical Microbiology and Infectious Disease 22, 54 - 57. 

Shaw, E. I., Dooley, C. A., Fischer, E. R., Scidmore, M. A., Fields, K. A. & Hackstadt, T. (2000). Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycle. Molecular Microbiology 37, 913 - 925.

Shen, L., Shi, Y., Douglas, A. L., Hatch, T. P., O'Connell, C. M., Chen, J. M. & Zhang, Y. X. (2000). Identification and characterization of promoters regulating tuf expression in Chlamydia trachomatis serovar F. Archives of Biochemistry and Biophysics 379, 46 - 56.

Solomon, A., Mabey, D., Holland, M., Alexander, N., Aguirre, A., Massae, P., Bailey, R., West, S., Foster, A. (2002). Quantification of nasal Chlamydia trachomatis infection in a trachoma endemic area of Tanzania. In Schachter et al., (eds) Chlamydial infections. Proceedings of the tenth international symposium on human chlamydial infections, pp 527 - 530. Published by International Chlamydia Symposium, San Francisco, CA 94110. ISBN 0-9664383-1-0 [See next].

Solomon, A. W., Holland, M. J., Burton, M. J., West, S. K., Alexander, N. D., Aguirre, A., et al., (2003). Strategies for control of trachoma: observational study with quantitative PCR. Lancet 362, 198 - 204. Full article

Tondella, M. L., Talkington, D. F., Holloway, B. P., Dowell, S. F., Cowley, K., Soriano-Gabarro, M., Elkind, M. S. & Fields, B. S. (2002). Development and evaluation of real-time PCR-based fluorescence assays for detection of Chlamydia pneumoniae. Journal of Clinical Microbiology 40,  575 - 583. Full article   

Welti, M., Jaton, K., Altwegg, M., Sahli, R., Wenger, A. & Bille, J. (2003). Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions. Diagnostic Microbiology and Infectious Diseases 45, 85 - 95.