TY - JOUR
T1 - Rational Design of CRISPR/Cas12a-RPA Based One-Pot COVID-19 Detection with Design of Experiments
AU - Malcı, Koray
AU - Walls, Laura E.
AU - Rios-solis, Leonardo
N1 - Funding Information:
This research was funded by the University of Edinburgh LMIC Partnerships Fund (PF_35) obtained by Dr. Leonardo Rios-Solis the YLSY program of the Ministry of National Education of Turkey the British Council (Grant Number: 527429894), and the Engineering and Physical Sciences Research Council (Grant Number: EP/R513209/1).
Funding Information:
This research was funded by the University of Edinburgh LMIC Partnerships Fund (PF_35) obtained by Dr. Leonardo Rios-Solis, the YLSY program of the Ministry of National Education of Turkey, the British Council (Grant Number: 527429894), and the Engineering and Physical Sciences Research Council (Grant Number: EP/R513209/1).
Publisher Copyright:
© 2022 American Chemical Society.
PY - 2022/4/15
Y1 - 2022/4/15
N2 - Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method’s sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.
AB - Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method’s sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.
KW - one-pot COVID-19 testing
KW - CRISPR/Cas12a
KW - molecular diagnosis
KW - definitive screening design
KW - reaction optimization
KW - recombinase polymerase amplification (RPA)
U2 - 10.1021/acssynbio.1c00617
DO - 10.1021/acssynbio.1c00617
M3 - Article
SN - 2161-5063
VL - 11
SP - 1555
EP - 1567
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 4
ER -