Is it important to use GAPDH in PCR to detect the pneumocystis jirovecii in BAL or sputum?
Dear Hosam Ghanem,
I am not really sure, if I understood your question, but I try to answer it. In order to detect a pathogen in a clinical (human) sample, you only need a specific PCR targeting one or several target genes or specific DNA sequences of the pathogen itself ("mono" or "multiplex" PCR).
PCR can be performed with a conventional thermocycler or with a a "real-time PCR" instrument allowing online -monitoring of the specific PCR product. Specificity of the PCR product has to be confirmed by specific hybridization (PCR, Realtime PCR) or sequencing of the amplicon(s).
In recent years additional PCR reactions were included in most diagnostic PCRs as external or internal controls for quality assurance. These additional reactions within your single reaction vial (then automatically resulting in an multiplex approach) are used to monitor proper DNA extraction and/or adequate sampling.
I anticipate that your question is focused on the later aspect: a specific GAPDH PCR targeting the DNA of the HOST (!), will allow you to ensure that your clinical sample is of human origin, is properly sampled from the patient (e. g. sputum, tracheal secret, BAL fluid) and that DNA extraction was efficient. Depending on the PCR approach, It may also allow for "quantification", also I feel the later issue is not very relevant.
Besides GAPDH (which is often used in pathology) other "house-keeping genes" of the host can be targeted (actin genes, etc.).
Conclusion: You may use a human-specific GAPDH PCR for monitoring the quality of your PCR reaction, DNA extraction and clinical sampling and you need addtional PCR protocols for your specific target pathogen (s).
For all targets ,PCR protocols are published or can be purchased commercially.
Best regards
Wolf Splettstoesser
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