Focus on Microbiology Research - (Series 2)

Viable PCR is a new technology that selectively amplifies the DNA of surviving cells. This allows researchers to quickly and reliably distinguish between surviving and dead cells without relying on time-consuming culture methods.

Defects of traditional PCR


A major drawback of traditional PCR methods is the inability to distinguish between living and dead microorganisms. This can lead to the generation of misleading results, such as overestimating the level of pollution. In addition, only for surviving microorganisms, traditional methods are not competent for accurate quantitative testing (Figure 3).

Figure 3. Traditional PCR techniques cannot distinguish between surviving and dead microbes. We prepared a mixed sample of heat inactivated and surviving Salmonella. The cells are lysed and the DNA is extracted. Run traditional real-time quantitative PCR assays. The difference in CT values ​​between different cell mixtures was not detected. This suggests that traditional PCR methods cannot distinguish between DNA from surviving and dead microorganisms.

PMA has efficient resolving power for surviving and dead cells

PMA selectively enters dead cells, which are activated by light to embed DNA and covalently bind to it, thereby strongly inhibiting the amplification reaction in subsequent PCR. The CT signal obtained from the dead cell DNA is significantly higher than the viable DNA, thereby achieving a clear distinction between viable and dead cells (Fig. 4).

Figure 4. PMA inhibits the amplification of dead cell DNA. Various concentrations of Salmonella typhimurium samples were prepared using buffered peptone water, and PMA treated and non-treated samples were prepared. In the experiment, it was found that the CT value of the PMA treatment group was significantly shifted upward compared with the untreated group, and the average value of ΔCT was about 11.4. This indicates that PMA inhibits further amplification of Salmonella dysentery DNA. No CT titration effect was observed in the Salmonella PMA treatment group as in the non-treated group, probably due to the lower copy number range in the sample due to the lower detection limit of the mericon Salmonella spp Kit used.

PMA selectively inhibits DNA detection in dead cells

When a mixed sample of viable and dead microorganisms is used, PMA can selectively inhibit the detection of dead cells without affecting the detection of viable cell DNA (Fig. 5). Dead cells in the sample do not affect the performance of the PMA. It is therefore believed that the results obtained accurately reflect the viable microorganisms in the sample.

Figure 5. Detection of dead cell DNA is still selectively inhibited in mixed samples of surviving and dead cells. The histogram on the left represents surviving Salmonella cells in buffered peptone water. The histogram on the right represents a sample of surviving and heat inactivated (dead) Salmonella mixed at different ratios of 10:90%, 50:50%, 90:10%, and 100:0%. The results demonstrated a high degree of agreement between the CT values ​​of the two groups (CT values ​​were less than 1). This confirms that PMA can effectively inhibit the detection of Salmonella in death, and that PMA can provide a high level of resolving power for mixed samples of surviving and dead bacteria.

PMA has efficient resolving power for viable and dead cells

The survival and dead cell resolving power of PMA ensures that you can easily and absolutely determine the effectiveness of your own sterilization control measures. When comparing the difference in CT values ​​(ΔCT) between the PMA-treated and the untreated group of the surviving and dead Salmonella mixed samples, we found that the maximum signal shift of the higher CT values ​​was around 15. This significant signal separation event can be used to clearly distinguish between PMA-bound dead cell DNA and PMA unbound viable cell DNA. This resolving power is still effective in the case where the proportion of viable cells in the sample changes little. This will ensure that you can measure the effectiveness of your own sterilization control measures easily and with absolute certainty.

Figure 6. PMA provides efficient survival and death cell differentiation. This shows the difference in CT values ​​between the PMA treated and non-treated groups in the mixed samples of surviving and dead Salmonella. Samples containing 100% Salmonella dying were up-regulated by up to about 15 CT values ​​under PMA treatment conditions compared to the untreated group. The PMA-mediated signal quenching effect is non-linear, and it is significantly increased in the detection of viable cells, and as a result, the CT value is significantly reduced relative to the untreated group, thereby increasing the global resolution sensitivity.

PMA technology for efficient detection in turbid media

One problem with the use of photoactive compounds is how to achieve cell viability assays in turbid media with less than ideal performance. Fortunately, the wavelengths used to activate PMA can penetrate turbid media.

We added control DNA (spiked-in) DNA to an analytical configuration for Listeria to ensure PMA reagent performance and proper operation. Comparison of control DNA CT values ​​between PMA treated and non-treated samples can be used to confirm the efficacy and correctness of the PMA reagent. Figure 7 shows the results of the PMA survival/death cell differentiation process, which includes the detection of target channels and control DNA (control channels) in a mixture of heat inactivated and surviving Listeria monocytogenes. In the target channel, there was no difference in CT values ​​between the PMA-treated and non-treated groups of Listeria monocytogenes samples, which is consistent with experimental expectations. The introduction of PMA treatment in a Listeria monocytogenes sample resulted in a expected increase in CT outcome values, which is a follow-up result of PMA's successful insertion into DNA (Figure 7A). In the control channel, the DNA incorporated into the Listeria samples showed a CT value movement expected to be around 6 and its range of motion was still within a specific 6 ± 2 interval, indicating that the result was not affected by the used Nutella ® Media Effects (Figure 7B).

These data clearly demonstrate that the activation intensity of PMA is sufficient to penetrate turbid samples and successfully mediate DNA insertion reactions.

Figure 7. Effect of homogeneous media on PMA survival/death cell differentiation. nA Inactivated or heat-inactivated Listeria is incorporated into a 2.5% Nutella matrix and processed separately. Listeria monocytogenes showed no change in CT values ​​between the PMA treated and non-treated groups. The PMA treatment group of the Listeria monocytogenes sample showed an expected shift in the CT signal, indicating successful completion of the embedded modification of the DNA. nB Add control DNA to surviving or dead cell samples containing the same 2.5% Nutella matrix. The background of surviving or dead cells did not significantly affect the detection of control DNA, and only the presence or absence of PMA was reflected in these results. The CT value movement around 6 still exists and meets the predetermined range of 6±2. These data suggest that the PMA activation intensity in this application is sufficient to penetrate the turbid sample and successfully mediate the DNA insertion reaction.

The above information comes from QIAGEN official website!


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