In this study, we demonstrated a specific, multiplex, PRI-lock probe-based, high-throughput detection assay using the Biotrove OpenArray™ platform for the detection and quantification of plant pathogens. The described application serves as a model for the development of rapid, molecular detection systems that offer an unprecedented combination of specificity, high throughput capabilities and robust target quantification.
To evaluate the designed PRI-lock probes and corresponding primer pairs, the probes were ligated on single target sequences and tested using conventional real-time PCR. In all cases, the targets were correctly detected and no false positives were observed, indicating highly specific ligation of the PRI-lock probes on their respective targets. Furthermore, a ligation reaction performed on non-target organisms with very similar ligation target sites did not result in detectable fluorescent signals. In agreement with the results obtained previously , we demonstrated that the PRI-lock probe system could discriminate non-target sequences on a single nucleotide difference. This is of prime importance, because non-target organisms with very similar ligation target sites might be present in diagnostic samples. In addition, the influence of multiple targets on PRI-lock probe based detection was tested, and it was demonstrated that the presence of multiple targets had no statistically significant influence. Our multiplex detection system provided truly independent detection of the different pathogens, with no evidence of inhibition due to possible ligation competition.
Quantitative diagnostic assays require a linear range of quantification of several orders of magnitude. Ligation of short oligonucleotides was previously used successfully for the characterization of gene expression and gene copy number [24, 25]. In these cases, however, the potential target concentration range was much lower. We showed that ligation of PRI-lock probes can reflect well the target quantity over at least 5 orders of magnitude.
The sensitivity of detection was determined by testing a 10-fold dilution series of ligation targets for all the PRI-lock probes. Sensitivities of 103 and 104 target copies/μl initial ligation mixture were achieved, depending on the PRI-lock probe. Assuming e.g. an average rRNA gene copy number of 200 times per fungal or oomycete genome, our assay would require between 50 and 500 target genomes per 10 μl initial ligation mixture for reliable detection. For organisms with lower rRNA copy numbers, such as bacteria, the assay would require more target genomes input compared to the previous example. However, the final input in the nanoliter real-time reactions is only a fraction of the original ligation mixture (< 0.1%). To increase the final assay detection sensitivity, not the ligation sensitivity but the copy number input of ligated PRI-locks per nanoliter reaction should be increased. Compared with other circularization probe-based assays, we already increased the applied PRI-lock probe concentration in our assay [21, 26, 27]. Increasing the PRI-lock probe concentration in the ligation mixture even further does not seem to yield further gains in sensitivity (not shown). A pre-amplification of the ligated PRI-lock probes therefore, could serve as an alternative strategy to increase the final assay sensitivity. PCR-based amplification however, often leads to amplification biases, changing the ratios between targets in the original biological sample . Non-PCR-based strategies, like linear amplification, have been shown to minimize amplification biases and generally conserve the target ratios originally present in the template mixture [29, 30]. Incorporating a strategy with a linear amplification of the ligated probes into the PRI-lock based detection assay, therefore, seems to represent a potential way of increasing detection sensitivity, without compromising quantitative power.
The target copy number used as template in the OpenArray™ is lower than the copy number in the conventional real-time PCR. Despite the lower copy number input, the observed CT values and the linear detection range of the developed PRI-lock system were about the same for the two real-time platforms (Figure 2B). It is known that, in nanoliter reactions, the concentration of the amplified products reaches the threshold concentration needed to provide a significant fluorescent signal sooner than in microliter reactions . This property was previously exploited to detect as little as one template molecule in nanoliter PCR amplifications, without the need to increase the PCR assay cycle number [19, 31]. Together with the improved CT calling algorithm of the Biotrove OpenArray™ platform , this results in a performance equal to conventional real-time PCR despite the reduction in template target copy number.
The robustness of the PRI-lock system was tested by analyzing the inter-array variation of the OpenArray™ platform and the assay-to-assay reproducibility of the PRI-lock probes. Both, the inter-array and assay-to-assay variation were very low, demonstrating the reproducibility and quantitative reliability of the PRI-lock detection assay. The developed quantitative multiplex detection assay was validated by testing various artificial mixtures of target DNAs. We demonstrated that the calculated number of P. infestans and E. carotovora carotovora input targets, based on the observed CT values and the corresponding calibration formulas, were highly similar to the actual P. infestans and E. carotovora carotovora input target numbers. In a further test, P. infestans and E. carotovora carotovora targets were mixed in different ratios, using target concentrations within the linear detection range of the entire probe set. For both targets, a reciprocal dynamic range of 104 was established in a background of 20 ng non-target DNA. The observed dynamic range is an improvement over most previously developed multiplex pathogen detection assays, where the dynamic range was often limited to 100–1000 [21, 32]. In the Biotrove OpenArray™ platform, target PCR amplifications are completely independent of each other, and, consequently, no PCR competition among the different ligated PRI-lock occurs. The dynamic range of detection in the PRI-lock detection assay is therefore, as long as the PRI-lock probes ligate on their respective targets, practically unlimited. Finally, artificial mixtures of genomic DNAs in different concentration ratios were tested. The components of the pathogen mixture were identified, and the original target input was calculated using the calibration formulas. Moreover, the ratios of the targets among the different ligation samples were correctly identified as well. The consistent, 10-fold discrepancy in observed target amounts between the E. carotovora carotovora and G. Proteo bacterial PRI-lock probes is a consequence of the different ligation regions for each probe within the genomic DNA of E. carotovora carotovora. Apparently, the copy number of the 16S rRNA gene detected by the G. Proteo bacterial PRI-lock is approximately 10-fold higher than the recA gene detected by the E. carotovora carotovora PRI-lock probe.
Circularization probes have previously been applied successfully for the detection of multiple plant pathogens in diagnostic samples , but without the ability to quantify target numbers. To our knowledge, this report presents the first time that numerous plant pathogens could be simultaneously and accurately quantified using specific circularization probes in a single assay. For future applications, however, higher multiplexing is intended and therefore, the number of PRI-lock probes will be increased. Currently, assay background is considered as the biggest obstacle for increasing the level of multiplexing in traditional circularization probe-based diagnostic assays. Traditional circularization probes contain generic primer sites for PCR amplification . Multiplex PCR via general primer sites carries the potential for competition during amplification, with a cumulative increase in background, which reduces assay sensitivity. In contrast, each PRI-lock probe carries a unique pair of primer binding sites, unrelated to the sequences of all the other probes. Circularized PRI-lock probes can therefore be amplified individually. Increasing the level of multiplexing would not be expected to increase the background signal. To further guarantee low background, even in highly multiplex settings, we are currently developing a universal TaqMan® probe which hybridizes to the generic sequence incorporated in all the PRI-lock probes. In addition, including a universal TaqMan® probe should speed up data analysis, and therefore sample throughput, since there is no need to conduct amplicon dissociation curve analysis. Given the independent PCR amplification of the ligated PRI-lock probes and the three slide capacity of the OpenArray™ NT Cycler, it should be feasible to engineer ultra-high throughput arrays for the quantitative detection of hundreds of targets simultaneously.