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Subramaniam Sriram, Asa Ljunggren-Rose, Song-Yi Yao, William O. Whetsell, Reply to Hammerschlag et al., The Journal of Infectious Diseases, Volume 192, Issue 7, 1 October 2005, Pages 1307–1308, https://doi.org/10.1086/466538
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To the Editor—In their letter, Hammerschlag et al. [1] raise 2 major issues to which we would like to respond. These issues are the specificity of the polymerase chain reaction (PCR) assays and of the immunohistochemical (IHC) staining we used in our study [2]
The comments pertaining to the lack of specificity of the primer set and PCR amplification procedure for amplification of the major outer membrane protein (MOMP) gene are identical to those the same authors have raised previously; we have addressed them in the past [3]. There is no single accepted method for detection of Chlamydia pneumoniae DNA and, therefore, in-house assays that are verified internally are being used. We recognize the problems inherent in using nested PCR assays. To increase the specificity of our nucleic acid–based amplification assays, we required amplification with primers for 2 different C. pneumoniae genes to designate a sample as positive [4]
We must also point out that the conditions of our touchdown technique for the amplification of the 16s RNA gene began with an annealing temperature of 60°C—and not 58°C, as the authors indicated—a temperature that was similar to the beginning temperature (62°C) in experiments published by the authors and not 65°C, as they now suggest [5]
