POCT Detection of 14 Respiratory Viruses Using Multiplex RT-PCR

Ahn, Jeong Jin; Kim, Seung Jun; Yu, So Yeon; Koh, Eun Jung; Kim, Seunghwan; Sung, Heung Sup; Huh, Jin Won; Hwang, Seung Yong

doi:10.1007/s13206-021-00037-w

Cited by 20 publications

(18 citation statements)

References 30 publications

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“…This intrinsic activity of Cas12a or Cas13a in combination with various nucleic acid amplification techniques has enabled the development of reliable nucleic acid detection systems, such as an one-hour lowcost multipurpose highly efficient system (HOLMES) [14], DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) [10,15] and Specific High-sensitivity Enzymatic Reporter unlocking (SHERLOCK) [16,17]. As compared to traditional ones relying on nucleic acid amplification only [18][19][20], CRISPR/Cas-based diagnostic systems exhibit better sensitivity and selectivity due to the highly efficient catalytic activity of Cas proteins.…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

et al. 2022

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In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5′-TTATT-CCCCC-3′; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5′-TTATT-3′) used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin ( stn ) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems. Supplementary Information The online version contains supplementary material available at 10.1007/s13206-022-00081-0.

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Section: Introductionmentioning

confidence: 99%

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

et al. 2022

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“…STAT-DNA is a nanoparticle-based and simple visual detection system for amplified DNA without any substantial requirements, such as a centrifugal device, gel column or membrane for separation, or fluorescent detection [ 46 ]. The detection sensitivity of the STAT-DNA system was comparable to that of the conventional clinical-grade RT-PCR analysis.…”

Section: Discussionmentioning

confidence: 99%

Nanoparticle-Based Visual Detection of Amplified DNA for Diagnosis of Hepatitis C Virus

Kim

et al. 2022

Biosensors

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Rapid, simple, and inexpensive diagnostic point-of-care tests (POCTs) are essential for controlling infectious diseases in resource-limited settings. In this study, we developed a new detection system based on nanoparticle–DNA aggregation (STat aggregation of tagged DNA, STAT-DNA) to yield a visual change that can be easily detected by the naked eye. This simplified optical detection system was applied to detect hepatitis C virus (HCV). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers labeled with biotin and digoxigenin. Streptavidin-coated magnetic particles (1 μm) and anti-digoxigenin antibody-coated polystyrene particles (250–350 nm) were added to form aggregates. The limit of detection (LoD) and analytical specificity were analyzed. The STAT-DNA results were compared with those of the standard real-time PCR assay using serum samples from 54 patients with hepatitis C. We achieved visualization of amplified DNA with the naked eye by adding nanoparticles to the PCR mixture without employing centrifugal force, probe addition, incubation, or dilution. The LoD of STAT-DNA was at least 101 IU/mL. STAT-DNA did not show cross-reactivity with eight viral pathogens. The detection using STAT-DNA was consistent with that using standard real-time PCR.

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“…In fact, many existing SARS-CoV-2 diagnostic testing assays either detect multiple fragments from one gene or multiple genes to reduce false negative rates due to RNA degradation or false positive rates due to amplification errors [ 8 , 9 , 10 , 11 , 12 ]. Many researchers have also developed multiplexed virus diagnostic testing assays that can detect SARS-CoV-2 variants and other respiratory viruses [ 13 , 14 , 15 , 16 , 17 ]. Some researchers have further developed multiplexed virus diagnostic testing platforms that are amenable for point-of-care (POC) use [ 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

et al. 2022

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The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.

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POCT Detection of 14 Respiratory Viruses Using Multiplex RT-PCR

Cited by 20 publications

References 30 publications

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Nanoparticle-Based Visual Detection of Amplified DNA for Diagnosis of Hepatitis C Virus

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

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