Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

BACKGROUND: Detection of bacteria by PCR applied for screening blood and blood products with special attention platelet concentrates. For practical use is expected that the detection system including Gram-positive, Gram-negative and Gram-bacterial non-stainable. It is quite challenging to achieve high sensitivity with clear negative controls with PCR reagents, due mainly Taq DNA polymerase is contaminated with traces of bacteria.

METHODS: Bacterial decontamination of Taq DNA polymerase tested by two different methods using restriction enzymes Sau 3A1 and microfiltration. Besides the commercially available Taq polymerase depleted bacterial DNA was included.A published real-time PCR specific for Gram-negative bacteria adapted to Gram-positive bacteria, including Staphylococcus species specific and Mycobacteria, and is used to fill three Taq polymer-ases discharged from bacteria

RESULTS DNA contamination: Despite published reports of successful DNA decontamination , all three approaches perform poorly in experiments conducted in this study. Sensitivity ranges from about 50-100 colony units (CFU) per PCR reaction to Escherichia coli and Staphylococcus epidermidis, corresponding to 1.250 to 2.500 CFU / ml sample material. Conclusion: It seems unsatisfactory to receive a high detection limit for the PCR bacteria even if highly multiplexed diagnostics. a reliable method for the decontamination of Taq DNA polymerase is required and will present one important step towards the detection of bacterial DNA with high sensitivity.


DNA analysis is the main procedure in genetic engineering. This time the analysis is often done by PCR with Taq DNA polymerase. Although the price of the last enzyme is quite low, the demand for analysis of various results in a lot of spending money that is not affordable for many laboratories. Meanwhile, many screening duties do not require highly purified enzyme. Taking into account the unique nature of the enzyme that allows for little simplifies the production without the use of expensive techniques such as column chromatography or the length and / or dialysis.

The following data routine use of Taq DNA polymerase is prepared in accordance with the protocol developed in our laboratory are presented. protocols only take a few hours to realize and does not need to be a qualified technician or costly equipment. But give an enzyme preparation suitable for most screening purposes. Taq DNA polymerase isolated stock can be stored as ammonium sulfate suspension in the refrigerator for a long time, not less than 6 months. Working enzyme solution prepared from stock suspension on demand, no more than once a month and can be stored well in the refrigerator.

 Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.
Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

Selection of Taq DNA polymerase efficiently to optimize the T-DNA genotyping method for the rapid detection of mutant Arabidopsis thaliana plants.

Port plant homolog of various animal genes involved in the metabolism of phosphorus, telomere biology and other cellular processes. Compared to experiment with many other multicellular organisms, research in the model plant Arabidopsis thaliana take advantage of a short generation time and increasing warehouse of genetic tools and GMOs, including a large collection of T-DNA knockout and line activation.

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Available to the public availability of thousands of transgenic Arabidopsis lines provide a unique opportunity to address a number of important biological questions. However, the identification of T-DNA mutant of an individual plant seedlings provided by the distribution centers stock biological remains laborious and time-consuming procedure.

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