The RT-QuIC is a rapid prion detection assay that is more amenable to high-throughput applications than the original QuIC and much less prone to generate spontaneous, unseeded positive reactions than the ASA assay. The sensitivity of the RT-QuIC is similar to the in vivo bioassay in hamsters, but is roughly 50–200 times faster and much less expensive.
Using this assay, we have been able to rapidly detect and quantify prion seeding activities in nasal lavages from clinically TME-affected hamsters. Considering that nasal lavages are likely to dilute endogenous nasal cavity fluids by at least 100-fold, these results confirm and extend a previous report of substantial prion infectivity in nasal secretions from hamsters in the clinical phase of HY TME infection 
. In the previous report, we detected nasal fluid prions by bioassay and the original immunoblot-based QuIC assay. In the current study, our ability to rapidly detect and quantitate prion seeding activity in nasal lavages using the RT-QuIC raises the possibility that such testing of nasal lavages or swabs could help in diagnosing prion disease infections of humans and animals on a high-throughput basis.
Our detection and quantitation of prion seeding activity in the CSF of 263K scrapie-infected hamsters suggests that CSF, being a relatively accessible specimen, should be collected for prion disease diagnosis by RT-QuIC. Interestingly, the CSF SD50 levels (105.7 and 104.6/ml) () were similar to the highest value obtained for nasal lavages (105.7/ml) (). However, the CSF should have at least 100-fold lower levels of prion seeding activity than the endogenous nasal fluids, given the considerable dilution that occurs when the nasal cavities are flushed with lavage buffer.
The origin of the rare positives that we observed in negative control RT-QuIC reactions () is difficult to ascertain. Because we simultaneously tested both positive and negative controls on the same plates, there was some, but obviously very low, potential for prion seeds to be inadvertently transferred from prion-seeded wells to adjacent negative control reactions. Moreover, given the very high sensitivity of the assay, even a minute contamination could elicit a false-positive reaction. Yet another explanation could be a cross contamination due to a failure of our plate sealer tape during the course of the reaction incubation. Fortunately, whether due to contamination or spontaneous amyloidogenesis, such apparent false positives are extremely rare and can simply be retested for confirmation.
It is likely that the same multimeric particles of abnormal PrP that stimulate conversion of PrPC or rPrPc to an abnormally folded form in in vitro reactions also cause prion “infections” in vivo. Consistent with this idea, we found that positive RT-QuIC reactions were obtained only with seeds derived from TSE-infected animals (except for the rare exceptions described above). Moreover, we obtained similar end-point dilutions of scrapie BH with both the bioassay and the RT-QuIC (). These results gave the appearance of a direct quantitative correspondence between the activities measured in these assays. Indeed, we expect that for prions of a particular strain and tissue source, there will be a proportional relationship between the activities measured by end-point dilution analyses with the RT-QuIC and animal bioassay. However, the sensitivities of these distinct assays will likely be influenced by some fundamentally different factors in vitro and in vivo and should not be expected to coincide as closely as they have in with all types of prion samples or all permutations of the assays. Indeed, further studies will be required to determine whether RT-QuIC assays detect naturally occurring PrP aggregates that are associated with familial PrP mutations and disease, but are non-infectious in bioassays. This anticipated variability of the RT-QuIC and bioassay with different prion sample types does not diminish the utility of the RT-QuIC in assessing the relative amount of prion seeding activity in samples of similar nature. In further developments of RT-QuIC assays for certain purposes, e.g. diagnostic testing, the possibility that certain abnormal non-PrP amyloids could give false positive RT-QuIC reactions should also be considered.
The end-point dilution strategy for determining relative seed concentrations should be applicable to amyloid seeding assays for a variety of misfolded protein aggregates regardless of the means of detecting the amyloid product, e.g. by ThT fluorescence as in the ASA 
and RT-QuIC assays, or immunoblotting as in PMCA 
, rPrP-PMCA 
or original QuIC 
assays. Like the RT-QuIC, many amyloid-seeded polymerization reactions progress rapidly to completion after a lag phase, providing an all-or-nothing response within appropriately selected time frames. This typical feature of seeded polymerization reactions should facilitate determinations of the proportion of positive reactions among replicates at a given sample dilution. Analyses of data from serial dilutions of various samples using the Spearman-Kärber 
or Reed-Muench 
algorithms can improve estimates of SD50
values per unit volume, which then indicate the relative concentrations of seeding activity in the samples.
As noted above, Chen and colleagues have recently described an alternative means of obtaining quantitative estimates of prion seeding activity using PMCA reactions, called qPMCA 
. Rather than assaying serial dilutions of a sample and determining the end point dilution, as we demonstrate here, a single sample dilution is assayed in serial PMCA reactions and the relative seeding activity is estimated from the number of serial PMCA rounds that are required to detect a positive response. The accuracy of qPMCA therefore depends on the strength of the inverse correlation between the prion seed concentration and number of rounds required. Although these investigators have documented such a correlation, its biochemical/kinetic basis remains unclear. In contrast, end-point dilution analyses can simply be explained as a titration of the active species to the detection limit. Further studies will be required to determine which approach to estimating relative prion concentrations is more robust and practical for comparing specific sample types.
Within individual RT-QuIC experiments composed of multiple, simultaneous reactions, we observed a clear dependence of the lag phase on the concentration of seed, as illustrated in . The lag phase might be considered analogous to the TSE incubation period between the inoculation and the near terminal stage of disease. In certain combinations of host and TSE strain, standard curves correlating bioassay incubation period with inoculated dose can be established and used to determine relative prion infectivity levels in unknown samples without resorting to more time-consuming and animal-intensive end-point dilution analyses. An analogous correlation between prion seed concentration and lag phase in the seeding assays like the RT-QuIC or ASA might also allow for seeding activity estimation without testing serial dilutions of each unknown. However, further work will be required to determine the efficacy, reproducibility, and validity of such an approach. In the mean time, the end-point dilution approach described in the current manuscript provides a clear means of quantitating prion seeding activity.
In summary, the end-point dilution RT-QuIC analysis provides quantitative comparisons of prion seeding activity. Although the extent to which prion seeding activity correlates quantitatively with infectivity in vivo under various other circumstances remains to be determined, we have shown that the RT-QuIC assay provides rapid and highly sensitive discrimination of prion-infected and uninfected brain tissues, nasal lavages, and CSF.