Journal of Clinical Microbiology, March 2005, p. 1492-1493, Vol. 43, No. 3
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.3.1492-1493.2005
| LETTER TO THE EDITOR |
Amplification of Coccidioidal DNA in Clinical Specimens by PCR
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In the May 2004 issue of the Journal of Clinical Microbiology, Johnson et al. described a PCR assay for detection and identification of coccidioidal DNA in clinical specimens (2). The PCR products amplified from murine and human serum samples were specified solely by agarose gel electrophoresis in comparison to a sequenced product obtained from cloned DNA of a lab strain of Coccidioides immitis.
A variation of up to 20 bp (10%) can be expected when the lengths of fragments 200 to 400 bp in size are determined by agarose gel analysis. High-resolution agarose might reduce the size variation to less than 2%, but it was not used in the study. Electrophoresis of the PCR products through the use of a denaturing polyacrylamide gel would have been appropriate to determine the DNA size. However, according to published quality standards (3, 4) a positive result of a nucleic acid amplification technique (NAT) has to be confirmed by hybridization, restriction enzyme analysis, or sequencing: "Length of the amplified product after separation by electrophoresis is insufficient for interpreting the results of a simple NAT" (4).
The authors describe the primers of the PCR assay as specific for coccidioidal DNA according to a reference (1). In their excellent work Greene et al. used the primers to amplify the internal transcribed spacer (ITS) region of cultures of Coccidioides isolated from mice infected with soil samples. They tested DNA extracted from cultures and identified each PCR product by sequencing! By using DNA from related fungi they proved their PCR assay to be specific for Coccidioides spp. Before using the PCR assay in clinical samples it is necessary to exclude amplification of human and murine DNA by the primers. A BLAST search in GenBank reveals that the primers show homology to fungal genes of only Coccidioides spp. However, identity of up to 18 of the 20 bases of each primer with those of several human and murine genes was also detected. The homology was found especially at the 3' endings of both primers responsible for polymerase binding. Thus, unspecific or cross-amplifications are very likely to occur when human or murine DNA is used as a template. Johnson et al. found a PCR product in only a few samples. However, they failed to demonstrate the presence of amplifiable human or murine DNA in the other samples by a control PCR targeting a murine or human gene. Thus, the possibility that negative PCR results were due to absence of human or murine DNA cannot be excluded.
In conclusion, Johnson and coworkers have to use appropriate standard methods to prove the specificity of their PCR products obtained from clinical specimens before discussing their findings and before the assay can be recommended for diagnostic purposes.
To maintain the high standards of the Journal of Clinical Microbiology, manuscripts on new diagnostic PCR assays for agents of infectious diseases should only be published if the PCR products amplified from clinical specimens have been identified by sequencing.
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TopLetterReferencesLetter |
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- Greene, D. R., G. Koenig, M. C. Fisher, and J. W. Taylor. 2000. Soil isolation and molecular identification of Coccidioides immitis. Mycologia 92:406-410.[CrossRef]
- Johnson, S. M., K. A. Simmons, and D. Papagianis. 2004. Amplification of coccidioidal DNA in clinical specimens by PCR. J. Clin. Microbiol. 42:1982-1985.
[Abstract/Free Full Text] - National Committee for Clinical Laboratory Standards. 1995. Molecular diagnostic methods for infectious diseases, approved guideline. Document MM3-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- Roth, A., H. Mauch, and U. Göbel. 1997. Nucleic acid amplification techniques. MiQ 1. Quality standards for microbiological diagnostic techniques for infectious diseases. Gustav Fischer, Stuttgart, Germany.
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Ralf Bialek*
Institute for Tropical Medicine University Hospital Tübingen Keplerstrasse 15 72074 Tübingen, Germany
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| * Phone: 49 7071 2982367,Fax: 49 7071 29 5267,E-mail: ralf.bialek{at}med.uni-tuebingen.de |
Authors’ Reply
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Thank you for the opportunity to respond to the letter submitted by Dr. Bialek to the Journal of Clinical Microbiology regarding our article "Amplification of Coccidioidal DNA in Clinical Specimens by PCR."
While the purpose of the letter appears to suggest a standard for publication, i.e., the sequencing of all PCR products, it represents a misunderstanding of the published material.
Point 1.Dr. Bialek states that "In their excellent work Greene et al. used the primers to amplify the internal transcribed spacer (ITS) region of cultures of Coccidioides isolated from mice infected with soil samples." While Dr. Bialek is correct that Greene et al. amplified DNA from cultures of Coccidioides isolated from soil, this material was at no time used to infect mice.
Point 2.Dr. Bialek states that "The PCR products amplified from murine and human serum samples were specified solely by agarose gel electrophoresis in comparison to a sequenced product obtained from cloned DNA of a lab strain of Coccidioides immitis." This statement is partially correct. Products of PCR amplification of human and mouse serum were subjected to agarose gel electrophoresis. However, the products were compared with products of a positive-control reaction mixture, amplified concurrently, that contained added coccidioidal genomic DNA from C. immitis strain Silveira.
Point 3.Dr. Bialek suggests that none of the PCR products amplified from the clinical samples were sequenced. While most were not, one was. Perhaps the origin of this sequenced product was unclear, leading Dr. Bialek to presume that the "positive product" that was discussed was a positive-control product. This product was obtained following amplification of material isolated from serum. The product was cloned, and the plasmid was isolated and sequenced.
Point 4.Dr. Bialek states that the primers have some identity to several human and murine genes and that unspecific or cross-amplification might occur with human or murine DNA. A BLAST search of the National Center for Biotechnology Information database did indeed indicate areas on multiple murine chromosomes that were homologous to the individual primers. However, the presence of these sequences does not necessarily predict amplification. To amplify a portion of DNA, both primers are required to anneal to a given sequence. Additionally, for this assay, they must yield a product that is comparable in size to a product from a control reaction. If the product can have a size variation of 20 bp (10%) as Dr. Bialek states, then the nonspecific reaction would need to yield a product of between 220 and 260 bp. A BLAST search using both primers that were used for amplification in the assay (ITS C1A and ITS C2) failed to yield any significant matches. If Dr. Bialek had indicated which sequences might amplify with the primers in question, a more comprehensive response could be made regarding this speculation.
Point 5.Dr. Bialek suggests that the amplified products obtained were a result of unspecific or cross-amplification of human or murine DNA that was present in the serum samples. Dr. Bialek further suggests that the nonappearance of an amplified product (the majority of samples evaluated) was attributable to the absence of human or murine DNA. It seems unlikely that one of multiple murine serum samples collected would contain murine DNA while all the remaining samples, which were handled similarly, would not.
We hope that the above discussion clarifies interpretation of the results we presented.
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Suzanne M. Johnson* Keira A. Pappagianis Department of Medical Microbiology and Immunology School of Medicine University of California, Davis Davis, CA 95616
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| * Phone: (530) 752-7214, Fax: (530) 752-8692, E-mail: smjohnson{at}ucdavis.edu |
Journal of Clinical Microbiology, March 2005, p. 1492-1493, Vol. 43, No. 3
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.3.1492-1493.2005
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