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Journal of Clinical Microbiology, November 2002, p. 4393, Vol. 40, No. 11
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.11.4393.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
| LETTER TO THE EDITOR |
High Frequency of Competitive Inhibition in the Roche Cobas AMPLICOR Multiplex PCR for Chlamydia trachomatis and Neisseria gonorrhoeae
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The term multiplex PCR refers to simultaneous amplification of more than one target in a single PCR. This method has some advantages but presents the possibility of competition between multiple targets for a finite number of reagents, which may invalidate the assay.
The Roche Cobas AMPLICOR multiplex PCR for Chlamydia trachomatis and Neisseria gonorrhoeae, a U.S. Food and Drug Administration-approved assay, can detect both C. trachomatis and N. gonorrhoeae from a single specimen. PCR amplification of C. trachomatis and N. gonorrhoeae proceeds in one tube with a shared enzyme and shared nucleotides but independent biotinylated primers. An optional internal control (IC) permits detection of amplification inhibition. The IC DNA has primer binding sequences identical to those of the C. trachomatis target. Detection is accomplished by using oligonucleotide probes that are unique for C. trachomatis, N. gonorrhoeae, and IC, respectively, with colorimetric quantification by spectophotometer. A negative assay result is valid if the IC optical density (OD) is 
0.2, indicating successful amplification (Method Manual, Cobas Amplicor, Roche Diagnostics, 12/1999, Revision 3.0).
We performed 580 multiplex PCRs on endocervical and urethral swab specimens, and 58 assays yielded positive results for C. trachomatis alone (OD 2.0), 13 assays yielded positive results for N. gonorrhoeae alone (OD 3.5), and 4 assays yielded positive results for both. Of the 58 assays positive for C. trachomatis, 13 (22.4%) had IC OD values that were <0.2 (mean OD, 0.059). The results of these 13 assays were correctly interpreted as C. trachomatis positive (mean C. trachomatis OD, 3.187). Because of the failure of the IC to amplify, the N. gonorrhoeae OD values for these 13 were invalid. The limiting reagent could be the primer, shared by C. trachomatis and IC. In the absence of IC amplification, it was not clear that there were a sufficient number of reagents, aside from the primer, to amplify N. gonorrhoeae. Of these 13 assays, 11 had OD values interpreted as negative for N. gonorrhoeae (mean N. gonorrhoeae OD, 0.047). Five of these specimens from the 11 assays were cultured and yielded negative results. Two additional specimens that turned out to be N. gonorrhoeae culture positive had equivocal N. gonorrhoeae OD values of 2.013 and 3.492, respectively, resolvable as positive by duplicate repeat testing.
A Roche Molecular Systems study suggested that competitive inhibition occurs when the relative concentration of one target is extremely high and that the competition is for reagents other than the primer. This Roche paper discusses the optional use of the IC for increased sensitivity as well as retesting of existing specimens to eliminate nonspecific, labile polymerase inhibition, which we saw in five specimens not discussed here (1). However, we had a very high rate of competitive inhibition not correctable by repetition. Experimental dilution of the specimens did result in IC amplification, but also, in one case, converted a positive C. trachomatis assay result to negative.
The Molecular Pathology Checklist of the College of American Pathologists states that "the laboratory should be able to distinguish a true negative result from a false negative result due to PCR failure." ICs accomplish this. The competitive inhibition described could be resolved by repeat amplification with only the N. gonorrhoeae primer. For the two equivocal specimens, positive for C. trachomatis, the initial OD value cutoff for N. gonorrhoeae positivity could be lower than the stated 3.5; perhaps 2.0 could be used as the cutoff OD value for duplicate repeat testing of specimens that produce equivocal results. Competitive inhibition is not fully addressed in the Method Manual. Undetected false negatives, due to failure to include the so-called optional IC, are misleading.
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- Rosenstraus, M., Z. Wang, S.-Y. Chang, D. DeBonville, and J. P. Spandoro. 1998. An internal control routine diagnostic PCR: design, properties, and effect on clinical performance. J. Clin. Microbiol. 36:191-197.
[Abstract/Free Full Text]
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Marilyn S. Hamilton* Mary Otto Angela Nickell David Abel Yolanda Ballam Virology Laboratory Department of Pathology
Robert Schremmer
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* Phone 816-234-3234 Fax: 816-802-1492 E-mail: mhamilton{at}cmh.edu. |
Journal of Clinical Microbiology, November 2002, p. 4393, Vol. 40, No. 11
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.11.4393.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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