We performed PCR with blood samples and blood culture bottles to correlate the significance of the sample type.
As a validation run, we used samples from K2-EDTA containers and blood culture bottles for a multiplex PCR run. The results show the expected results in K2-EDTA sample and there was no detection in the blood culture bottle sample, even Internal Control (IC) was not detected.
Please anyone expert in this field, suggest the reason for this failure and how we can overcome this issue.
I really appreciate any help you can provide.
Can you explain the purpose of the experiment and what exactly is in the samples? I'm not clear on whether you are trying to detect human genomic DNA, bacterial genomic DNA, or something else. Are you doing something to isolate the DNA first, or just taking the sample directly from the bottles?
Laura Leighton Thanks for the reply.
Yes, I will explain in detail.
1. The objective is to detect the bacterial genomic DNA of microbial infection in a patient using RT-PCR.
2. We have two sample-type sources, 1. ETDA sample 2. blood culture bottles. Both samples were collected at the same time from a patient.
3. We have a commercial multiplex kit for detecting genomic DNA in microbes. From the experiment, we observed favorable results in the EDTA sample and no detection in the blood culture bottle sample.
In other hand, the blood culture bottle was found to have the growth of several micro-organisms from the microbiology laboratory.
The query we have is
What is the reason for the failure of this run?
Whether any ingredients/composition in the blood culture bottle affect or inhibit the run?
At the same time, we are not sure whether blood culture bottles are recommended sample type for PCR detection.
Thank you
Are you trying to detect a specific pathogen? If your internal control did not work, it suggests you may have a PCR inhibition - make sure you use a DNA extraction kit that removes inhibitory molecules.
As for the different results, keep in mind that EDTA blood and blood culture are very different sample types - you will have a broader range of pathogens in EDTA blood but in lower concentrations, while in blood culture you will have 1-2 pathogens (typically) at higher concentrations.
Thanks for the reply Péter Gyarmati .
In this case, we try to detect multiple pathogens (~30 different pathogens) from a single sample.
From the results, we got IC detection in EDTA and no detection in blood culture. Based on your point, It can be pure PCR inhibition or extraction kit inefficiency.
Refer the following article:
Article Improved Amplification of Microbial DNA from Blood Cultures ...
In culture bottles, it contains Sodium Polyanethole Sulphonate (SPS) which acts as a PCR inhibitor. These SPS are soluble in water and insoluble in alcohol. This manuscript has a procedure "Benzyl alcohol guanidine hydrochloric organic method" which is helpful to remove the PCR inhibitors in a simple extraction protocol.
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