Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples

Haley, Nicholas J.

doi:10.1002/cpmb.118

Cited by 7 publications

(5 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Moreover, the high concentration of guanidine-HCl solution used as a denaturant often damages nickel resins, deteriorating their binding capacity even further. Thus, laboratories preparing recPrP for RT-QuIC treat the Ni-NTA resins as single-use (Haley, 2020).…”

Section: Discussionmentioning

confidence: 99%

Real‐time quaking‐induced conversion assay using a small‐scale substrate production workflow for the diagnosis of Creutzfeldt‐Jakob disease

Thanapornsangsuth

Chongsuksantikul

Saraya

et al. 2023

Journal of Neurochemistry

View full text Add to dashboard Cite

The lack of a dedicated surveillance program for prion disease, particularly in low‐ and middle‐income countries (LMICs), has hindered the global effort to address this public health threat. Although cerebrospinal fluid (CSF) Real‐time quaking‐induced conversion (RT‐QuIC) is considered the most reliable test for sporadic Creutzfeldt‐Jakob disease (sCJD), its availability in LMICs is limited because of its cost and technical difficulty in generating the recombinant prion protein substrate (recPrP). This study aimed to evaluate the performance of RT‐QuIC with recPrP produced in‐house through a small‐scale method—that is, the application of reusable prepacked chromatography columns and subsequent dialysis. Here, CSF specimens from patients suspected of having prion disease were consecutively collected and stored between October 2015 and January 2023. Electronic medical record data were reviewed to clinically classify participants as probable sCJD or non‐sCJD. CSF RT‐QuIC was performed using in‐house recPrP. Its specificity and sensitivity for diagnosing probable sCJD were reported, along with details of other clinical data and investigations. We found that among 39 eligible participants, with a median (interquartile range) age of 64 (56–70) years and 16 (41%) female, 13 had probable sCJD and the remaining 26 unequivocally suffered from nonprion disorders. Magnetic resonance imaging and electroencephalogram were suggestive of sCJD in 100% (13/13) and 46.2% (6/13) of sCJD participants, respectively. RT‐QuIC was positive in 12/13 sCJD participants (sensitivity 0.92, 95% confidence interval [CI] 0.67–0.99) and negative in all non‐sCJD participants (specificity 1.00, 95% CI 0.87–1.00). CSF tau/p‐tau ratio showed sensitivity and specificity of 0.62–1.0 and 0.85–1.0, respectively. In summary, RT‐QuIC using recPrP generated through a small‐scale workflow demonstrated great performance in detecting sCJD. Given its performance results along with its low cost, this technique could feasibly be implemented in LMICs and potentially be the first step toward establishing local prion disease surveillance programs.

show abstract

Section: Discussionmentioning

confidence: 99%

Real‐time quaking‐induced conversion assay using a small‐scale substrate production workflow for the diagnosis of Creutzfeldt‐Jakob disease

Thanapornsangsuth

Chongsuksantikul

Saraya

et al. 2023

Journal of Neurochemistry

View full text Add to dashboard Cite

show abstract

“…RT-QuIC assays were performed as previously described [ 22 ], with slight modifications. Briefly, 95 µL of the reaction master mix and 5 µL of diluted obex or RLN tissue homogenate were added to each well of a 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer

Yilmaz,

Morrill,

Pilot

et al. 2024

Veterinary Sciences

View full text Add to dashboard Cite

Real-time quaking-induced conversion (RT-QuIC) assays have become a common tool to detect chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for methods to optimize the RT-QuIC assay. In this study, receiver operating characteristic (ROC) analysis was applied to optimize the assay duration for CWD screening in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 33 h for obex and 30 h for RLN based on Tstdev, and 29 h for obex and 32 h for RLN based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. At a replicate level, using the optimized assay durations with TStdev and TMPR, the level of agreement with enzyme-linked immunosorbent assay (ELISA) was significantly higher (p < 0.05) than that when using a 40 h assay duration. These findings demonstrate that the optimization of assay duration via a ROC analysis can improve RT-QuIC assays for screening CWD in white-tailed deer.

show abstract

“…RT-QuIC assays were performed as previously described [21], with slight modifications. Briefly, 95 µL of the reaction master mix and 5 µL of diluted obex or RLN tissue homogenate were added to each well of a 96-well plate.…”

Section: Rt-quic Methodsmentioning

confidence: 99%

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer

Yilmaz,

Morrill,

Ward

et al. 2023

Preprint

View full text Add to dashboard Cite

Real-time quaking-induced conversion (RT-QuIC) assays have become common in the detection of chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for optimization of the RT-QuIC assay duration. In this study, receiver operating characteristic (ROC) analysis was applied to optimize assay duration for detection of CWD in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 27 h for both obex and RLN based on Tstdev, and 27 and 28 h for obex and RLN, respectively, based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. Results by RT-QuIC using optimized duration based on Tstdev and TMPR were in 100% and 92.3 % or higher agreement with those by the widely used screening assay, ELISA. In comparison, when using a 40 h assay duration, the agreement between RT-QuIC and ELISA reduced to 89.2% or higher, and the RT-QuIC results were significantly (p < 0.05) different from those using optimum durations. These findings demonstrated that the application of ROC analysis for the optimization of assay duration could improve the RT-QuIC assay for screening CWD in white-tailed deer.

show abstract

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples

Cited by 7 publications

References 50 publications

Real‐time quaking‐induced conversion assay using a small‐scale substrate production workflow for the diagnosis of Creutzfeldt‐Jakob disease

Real‐time quaking‐induced conversion assay using a small‐scale substrate production workflow for the diagnosis of Creutzfeldt‐Jakob disease

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer

Contact Info

Product

Resources

About