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  Tania Crucitti
  Published on: 13 May 2016
• Taq Polymerase vs. Ampli Taq Gold Polymerase for Trichomonas vaginalis PCR detection.
  Robert H. Gilman
  Published on: 13 May 2016

• Published on: 13 May 2016

  Author's reply

  • Tania Crucitti, Scientific collaborator
  • Other Contributors:
    • Eddy Van Dyck

  Dear Editor

  The purpose of our study was to evaluate five different primer sets described in the literature for the amplification of Trichomonas vaginalis. We therefore used the same working conditions for the five primers sets, i.e. the same extraction method, thermocycler, reagents etc. It was not our aim to re-validate these primer sets.

  For all of the five primer sets we used the AmpliTaq Gold poly...

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  Dear Editor

  The purpose of our study was to evaluate five different primer sets described in the literature for the amplification of Trichomonas vaginalis. We therefore used the same working conditions for the five primers sets, i.e. the same extraction method, thermocycler, reagents etc. It was not our aim to re-validate these primer sets.

  For all of the five primer sets we used the AmpliTaq Gold polymerase, which has some advantages compared to the simple Taq polymerase. The AmpliTaq Gold Polymerase is inactive at ambient temperatures, non-specific binding is avoided and preparation of reactions is facilitated. Activation of the AmpliTaq Gold Polymerase occurs only when heated before the thermal cycling, and is thus similar to a hot-start PCR. The AmpliTaq Gold Polymerase requires a pre-denaturation step at 95°C for 5 minutes, as shown in Table 2. The overall PCR product yield is also greater.[1]

  In our laboratory the AmpliTaq Gold Polymerase works very well. We follow strict quality guidelines, meaning that prior to the introduction of a new batch of AmpliTaq Gold Polymerase we test the quality of the batch with a panel of known positive and negative specimens. Variability in Taq polymerase performance based on batch, concentration, and supplier has been documented.[2] We detected variability in Taq polymerase quality based on differences between batches.

  References

  (1) T Moretti, B Koons, and B Budowle. Enhancement of PCR amplification yield and specificity using AmpliTaq Gold ® DNA polymerase. BioTechniques 1998; 25: 716-722.

  (2) KD Tyler, G Wang, SD Tyler, and WM Johnson. Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J Clin Microbiol 1997; 35:339-346.

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  Conflict of Interest:
  None declared.

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• Published on: 13 May 2016

  Taq Polymerase vs. Ampli Taq Gold Polymerase for Trichomonas vaginalis PCR detection.

  • Robert H. Gilman, Proffessor, Director of the Tropical Medicine Institute
  • Other Contributors:
    • Holger Mayta, Maritza Calderon

  Dear Editor

  The article by Crucitti et al.[1] evaluated five PCR techniques for Trichomonas vaginalis including the one published by our group (Mayta et al. [2]). The authors however did not follow the protocol we published and so got results that we consider to be erroneous.

  In our work we used simple Taq polymerase while Crucitti used Taq gold for this purpose. He did this without chang...

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  Dear Editor

  The article by Crucitti et al.[1] evaluated five PCR techniques for Trichomonas vaginalis including the one published by our group (Mayta et al. [2]). The authors however did not follow the protocol we published and so got results that we consider to be erroneous.

  In our work we used simple Taq polymerase while Crucitti used Taq gold for this purpose. He did this without changing or proving that the same cycle conditions will provide similar sensitivity. In our studies using the same cycles Taq gold is rarely positive and frequently gives negative results even when the culture is positive. In addition, they used a DNA extraction that was different from the one we used for our Trichomonas study.

  In the article by Crucitti et al. the primer set TV1/TV2 was found to be less sensitive presumably due to the use of an untried new protocol. We feel strongly that unless the authors follow the original protocol their results for the PCR sensitivity are not valid. In any comparative study of methods the published protocol should be followed and not modified without validation. This appears not to be done by Crucitti et al.

  References

  (1) T Crucitti, E Van Dyck, A Tehe, S Abdellati, B Vuylsteke, A Buve, and M Laga. Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens. Sex Transm Infect 2003; 79: 393-398.

  (2) Mayta H, Gilman RH, Calderon MM, et al. 18S Ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis. J Clin Microbiol 2000;38:2683–7.

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  Conflict of Interest:
  None declared.

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