Effect of heat inactivation and bulk lysis on real-time reverse transcription PCR detection of the SARS-COV-2: an experimental study

Leta, Dereje; Gutema, Gadissa; Hagos, Gebremedhin Gebremichael; Diriba, Regasa; Bulti, Gutema; Sura, Tolawak; Ayana, Desta; Chala, Dawit; Lenjiso, Boki; Bulti, Jaleta; Abdella, Saro; Tola, Habteyes Hailu

doi:10.1186/s13104-022-06184-z

Cited by 4 publications

(3 citation statements)

References 26 publications

(37 reference statements)

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“…The column-based DNA purification process used in this work still requires additional reagents and centrifugation. To simplify the detection workflow, thermal lysis has been developed and demonstrated as a useful lysis method in viral nucleic acid detection, − which was adopted to mimic the virus lysis and HPV DNA release as the sample preparation step. Then, the SPEEDi-CRISPR assay was utilized to capture target DNA directly from the serum samples without any DNA purification step.…”

Section: Resultsmentioning

confidence: 99%

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a

Tian,

Yan,

Zeng

2024

ACS Appl. Mater. Interfaces

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Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans-cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.

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Section: Resultsmentioning

confidence: 99%

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a

Tian,

Yan,

Zeng

2024

ACS Appl. Mater. Interfaces

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“…Moreover, following the COVID-19 pandemic, the importance of Ct values has been further emphasized, with many studies focusing on exploring the correlation between Ct values and disease severity and transmissibility [25,26], as well as the correlation between Ct values and epidemiological trends [21]. In addition to the content of the tested samples, Ct values are also influenced by factors such as the temperature and optical performance of the qPCR instrument itself [27], the use of reagent kits, sample collection time, primer specificity, amplification efficiency, sample inactivation temperature, and inactivation time [28].…”

Section: Of 22mentioning

confidence: 99%

Physical Simulation-Based Calibration for Quantitative Real-Time PCR

Zhu,

Liu,

Xiao

2024

Applied Sciences

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The fluorescence quantitative polymerase chain reaction (qPCR) instrument has been widely used in molecular biology applications, where the reliability of the qPCR performance directly affects the accuracy of its detection results. In this paper, an integrated, physics-based calibration device was developed to improve the accuracy and reliability of qPCR, realizing the calibration of qPCR instruments’ standard curve through physical simulations. With this calibration device, the collected temperature was used as the control signal to alter the fluorescence output, which allowed different probes to simulate the Ct values corresponding to samples with varying initial concentrations. The temperature and optical performance of this calibration device were tested, followed by a comparative analysis comparing the on-machine test results with standard substances to assess the linearity and uniformity of the Ct values of the measured qPCR instrument. It has been proven that this physical calibration device can effectively replace the biochemical standard substance to carry out comprehensive calibration of the temperature and optical parameters of the qPCR instrument and provide a more reliable method for the periodic calibration and quality control of the qPCR instrument. This contributes to the accuracy and reliability of fluorescence qPCR instruments in the field of molecular biology.

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“…The column-based DNA purification process used in this work requires multiple reagents and centrifugation. To simplify the detection workflow, thermal lysis that has been developed and demonstrated as a useful lysis method in viral nucleic acid detection [38][39][40][41] was adopted to mimic the virus lysis and HPV DNA release as the sample preparation step. Then the SPEEDi-CRISPR assay was utilized to capture target DNA directly from the serum samples without any DNA purification step.…”

Section: Detection Of Hpv In Spiked Samples Using the Speedi-crispr A...mentioning

confidence: 99%

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Dual-Functional CRISPR-Cas12a

Tian

Zeng

2023

Preprint

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Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advance in CRISPR-based biosensing promises the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. Currently prevailing strategy of developing CRISPR assays exploits only the non-specific trans-cleavage function of a CRISPR-Cas12a/Cas13a system for detection and combines it with an additional pre-amplification reaction to enhance the sensitivity. In contrast to this single-function strategy, here we present a new approach that collaboratively integrates the dual functions of CRISPR-Cas12a: sequence-specific binding and trans-cleavage activity. With this approach, we developed a POC nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection assay Integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with the fluorogenic detection by the activated Cas12a on beads. Our proof-of-concept study demonstrated that the SPEEDi-CRISPR assay affords an improved detection sensitivity for human papillomavirus (HPV)-18 with a LOD of 2.3 fM and excellent specificity to discriminate HPV-18 from HPV-16, Parvovirus B19, and scramble HPV-18. Furthermore, this robust assay was readily coupled with a portable smartphone-based fluorescence detector and a lateral flow assay for quantitative detection and visualized readout, respectively. Overall, these results should suggest that our dual-function strategy could pave a new way for developing the next-generation CRISPR diagnostics and that the SPEEDi-CRISPR assay provides a potentially useful tool for point-of-care testing.

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Effect of heat inactivation and bulk lysis on real-time reverse transcription PCR detection of the SARS-COV-2: an experimental study

Cited by 4 publications

References 26 publications

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a

Physical Simulation-Based Calibration for Quantitative Real-Time PCR

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Dual-Functional CRISPR-Cas12a

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