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.

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.

Reduce the reaction volume and increase the concentration of Taq DNA polymerase increases STR DNA profile results from the low source Template real world: telogen hair.

OBJECTIVE
The main method for the analysis of low template DNA (LTDNA) is known as a method of low copy number (LCN), which involves increasing the number of PCR cycles (typically 34). In common with other LTDNA method, characterized by an imbalance LCN profile allele, decline, and the drop outs which require interpretation of complex rules. They often require replicate PCR reactions to produce a “consensus” profile in special facilities. The ideal method for the analysis of LTDNA should raise the profile of the results without a high error rate and is accomplished using the standard amenities, in addition to the minimum cost.


METHOD
In this study, we present a comparison of four methods of variation for amplification of STR from LTDNA broadly used, the kit is available commercially (AmpFℓSTR (®) Profiler Plus (®)): the standard method, the standard method to post a PCR clean up, the method of the LCN, and the reaction volume decreases with increased concentrations of Taq DNA polymerase.


RESULTS
Using the telogen hair-LTDNA common source and reference DNA match, LCN method produces the highest number of appropriate and non-appropriate alleles (ie, down-in). For comparison, the reaction volume was reduced by an increase in Taq Polymerase produce fuller profile and corresponding DNA (all alleles combined) and less off-ladder alleles of a variety of input DNA. In addition, this method results in less non-concordant allele of the LCN and not more than for standard PCR, which indicates that it may be preferable to increase PCR cycles for analysis LTDNA, either with or without consensus profiles and statistical modeling.

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CONCLUSION

Overall, this study highlights the importance and benefits of optimizing PCR conditions and to develop improved laboratory method to amplify and analyze LTDNA.