Sofia Valla

Bio: Sofia Valla is an academic researcher from Ciba Specialty Chemicals. The author has an hindex of 1, co-authored 1 publications receiving 5 citations. Previous affiliations of Sofia Valla include National Scientific and Technical Research Council.

Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.

Abstract: We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Laboratory Diagnosis of SARS-CoV-2 Pneumonia.

Abstract: The emergence and rapid proliferation of Coronavirus Disease-2019, throughout the past year, has put an unprecedented strain on the global schema of health infrastructure and health economy. The time-sensitive agenda of identifying the virus in humans and delivering a vaccine to the public constituted an effort to flatten the statistical curve of viral spread as it grew exponentially. At the forefront of this effort was an exigency of developing rapid and accurate diagnostic strategies. These have emerged in various forms over the past year-each with strengths and weaknesses. To date, they fall into three categories: (1) those isolating and replicating viral RNA in patient samples from the respiratory tract (Nucleic Acid Amplification Tests; NAATs), (2) those detecting the presence of viral proteins (Rapid Antigen Tests; RATs) and serology-based exams identifying antibodies to the virus in whole blood and serum. The latter vary in their detection of immunoglobulins of known prevalence in early-stage and late-stage infection. With this review, we delineate the categories of testing measures developed to date, analyze the efficacy of collecting patient specimens from diverse regions of the respiratory tract, and present the up and coming technologies which have made pathogen identification easier and more accessible to the public.

Diagnostics of COVID-19 Based on CRISPR–Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update

Abstract: The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR–Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR–Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR–Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

Inconsistent Treatments of CRISPR Kinetics Impair Assessment of Its Diagnostic Potential

Abstract: Abstract The scientific and technological advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is one of the most exciting developments of the past decade, particularly in the field of gene editing. The technology has two essential components, (1) a guide RNA to match a targeted gene and (2) a CRISPR-associated protein (e.g. Cas 9, Cas12 or Cas13) that acts as an endonuclease to specifically cut DNA. This specificity and reconfigurable nature of CRISPR has also spurred intense academic and commercial interest in the development of CRISPR-based molecular diagnostics. CRISPR Cas12 and Cas13 orthologs are most commonly applied to diagnostics, and these cleave and become activated by DNA and RNA targets, respectively. Despite the intense research interest, the limits of detection (LoDs) and applications of CRISP-based diagnostics remain an open question. A major reason for this is that reports of kinetic rates have been widely inconsistent, and the vast majority of these reports contain gross errors including violations of basic conservation and kinetic rate laws. It is the intent of this Perspective to bring attention to these issues and to identify potential improvements in the manner in which CRISPR kinetic rates and assay LoDs are reported and compared. The CRISPR field would benefit from verifications of self-consistency of data, providing sufficient data for reproduction of experiments, and, in the case of reports of novel assay LoDs, concurrent reporting of the associated kinetic rate constants. The early development of CRISPR-based diagnostics calls for self-reflection and urges us to proceed with caution.

CRISPR-Cas Biochemistry and CRISPR-Based Molecular Diagnostics.

Abstract: Polymerase chain reaction (PCR)-based nucleic acid testing has played a critical role in disease diagnostics, pathogen surveillance, and many more. However, this method requires a long turnaround time, expensive equipment, and trained personnel, limiting its widespread availability and diagnostic capacity. On the other hand, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has recently demonstrated capability for nucleic acid detection with high sensitivity and specificity. CRISPR-mediated biosensing holds great promise for revolutionizing nucleic acid testing procedures and developing point-of-care diagnostics. This review focuses on recent developments in both fundamental CRISPR biochemistry and CRISPR-based nucleic acid detection techniques. Four ongoing research hotspots in molecular diagnostics-target preamplification-free detection, microRNA (miRNA) detection, non-nucleic-acid detection, and SARS-CoV-2 detection-are also covered.