DNA Nanosieve-Based Regenerative Electrochemical Biosensor Utilizing Nucleic Acid Flexibility for Accurate Allele Typing in Clinical Samples

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

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CRISPR is a useful biological tool for detecting nucleic acid of SARS-CoV-2 in human clinical samples

Biomedicine & Pharmacotherapy ◽

10.1016/j.biopha.2021.111772 ◽

2021 ◽

pp. 111772

Author(s):

Md. Rashidur Rahman ◽

Md. Amjad Hossain ◽

Md. Mozibullah ◽

Fateh Al Mujib ◽

Afrina Afrose ◽

...

Keyword(s):

Nucleic Acid ◽

Clinical Samples ◽

Biological Tool

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Rapid electrochemical detection of coronavirus SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-21121-7 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Thanyarat Chaibun ◽

Jiratchaya Puenpa ◽

Tatchanun Ngamdee ◽

Nimaradee Boonapatcharoen ◽

Pornpat Athamanolap ◽

...

Keyword(s):

Electrochemical Biosensor ◽

Rolling Circle Amplification ◽

Clinical Samples ◽

Rolling Circle ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Sandwich Hybridization ◽

Redox Active ◽

One Step ◽

The One

AbstractCoronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

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Application of green synthesized WO3-poly glutamic acid nanobiocomposite for early stage biosensing of breast cancer using electrochemical approach

Scientific Reports ◽

10.1038/s41598-021-03209-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hassan Nasrollahpour ◽

Abdolhossein Naseri ◽

Mohammad-Reza Rashidi ◽

Balal Khalilzadeh

Keyword(s):

Breast Cancer ◽

Glutamic Acid ◽

Electrochemical Biosensor ◽

Early Stage ◽

Clinical Samples ◽

Breast Cancer Patients ◽

Serum Samples ◽

Electrochemical Approach ◽

Her 2

AbstractBiopolymer films have drawn growing demand for their application in the point of care domain owing to their biocompatibility, eco-friendly, and eligibility for in vivo analyses. However, their poor conductivity restricts their sensitivity in diagnostics. For high-quality electrochemical biosensor monitoring, two vital factors to be greatly paid attention are the effective merge of amplification modifiers with transducing surface and the superior linking across the recognition interface. Here, we introduce an enzyme-free electrochemical biosensor based on electrosynthesized biocompatible WO3/poly glutamic acid nano-biocomposites to address the hardships specific to the analysis of circulating proteins clinical samples. In addition to its green synthesis route, the poor tendency of both components of the prepared nano-biocomposite to amine groups makes it excellent working in untreated biological samples with high contents of proteins. Several electrochemical and morphological investigations (SEM, EDX, and dot mapping) were fulfilled to gain a reliable and trustful standpoint of the framework. By using this nanobiosensor, the concentration of HER-2 was detectable as low as 1 fg mL−1 with a wide linear response between 1 ng mL−1 and 1 fg mL−1. Meanwhile, the protocol depicted ideal specificity, stability, and reproducibility for the detection of HER-2 protein in untreated serum samples of breast cancer patients.

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Comparative performance and cycle threshold values of 10 nucleic acid amplification tests for SARS-CoV-2 on clinical samples

PLoS ONE ◽

10.1371/journal.pone.0252757 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252757

Author(s):

Miyuki Mizoguchi ◽

Sohei Harada ◽

Koh Okamoto ◽

Yoshimi Higurashi ◽

Mahoko Ikeda ◽

...

Keyword(s):

Nucleic Acid ◽

Diagnostic Performance ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Nucleic Acid Amplification Tests ◽

Cycle Threshold ◽

E Gene ◽

Viral Copy Number ◽

Gene Test ◽

Positive Results

Background A number of nucleic acid amplification tests (NAATs) for SARS-CoV-2 with different reagents have been approved for clinical use in Japan. These include research kits approved under emergency use authorization through simplified process to stabilize the supply of the reagents. Although these research kits have been increasingly used in clinical practice, limited data is available for the diagnostic performance in clinical settings. Methods We compared sensitivity, specificity, and cycle threshold (Ct) values obtained by NAATs using 10 kits approved in Japan including eight kits those receiving emergency use authorization using 69 frozen-stored clinical samples including 23 positive samples with various Ct values and 46 negative samples. Results Viral copy number of the frozen-stored samples determined with LightMix E-gene test ranged from 0.6 to 84521.1 copies/μL. While no false-positive results were obtained by any of these tests (specificity: 100% [95% CI, 88.9%-100%]), sensitivity of the nine tests ranged from 68.2% [95% CI, 45.1%-86.1%] to 95.5% [95% CI, 77.2%-99.9%] using LightMix E-gene test as the gold standard. All tests showed positive results for all samples with ≥100 copies/μL. Significant difference of Ct values even among tests amplifying the same genetic region (N1-CDC, N2) was also observed. Conclusion Difference in the diagnostic performance was observed among NAATs approved in Japan. Regarding diagnostic kits for emerging infectious diseases, a system is needed to ensure both rapidity of reagent supply and accuracy of diagnosis. Ct values, which are sometimes regarded as a marker of infectivity, are not interchangeable when obtained by different assays.

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Retrospective Analysis of 2019-nCov-Infected Cases in Dongyang, Southeastern China

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2020/7056707 ◽

2020 ◽

Vol 2020 ◽

pp. 1-6

Author(s):

G. G. Li ◽

Z. Lv ◽

Y. S. Wang ◽

J. F. Li ◽

L. F. Feng ◽

...

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Clinical Samples ◽

Test Results ◽

Infected People ◽

Sporadic Cases ◽

Asymptomatic Individuals ◽

Novel Coronavirus ◽

Acid Test ◽

Negative Conversion

The 2019 novel coronavirus (2019-nCov) has caused increasing number of infected cases globally. This study was performed to analyze information regarding the transmission route and presence of viral nucleic acids on several clinical samples. Confirmed 2019-nCov-infected cases were identified in Dongyang and were treated according to guidelines for the diagnosis of 2019-nCov infection released by the National Health Commission. Information regarding the contacts that the infected people had was collected to determine whether it caused clustered cases. A series of successive nucleic acid examination of feces, oropharyngeal swabs, and sputum was also performed, and the results were analyzed. A total of 19 confirmed cases of 2019-nCov infection were identified in Dongyang, Zhejiang Province, China. Five cases showed severe symptoms, and the remaining ones showed mild manifestations. Ten cases infected from two asymptomatic individuals were clustered into two groups. Among 14 cases with consecutive nucleic acid test results, four patients showed positive results in feces after their negative conversion in oropharyngeal swabs. Asymptomatic individuals with the virus could cause 2019-nCov clustered cases, and the clustered cases may differ from sporadic cases on age and length of hospitalization. In addition, nucleic acids in feces last longer than those in oropharyngeal swabs.

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A novel electrochemical biosensor for ultrasensitive Hg2+ detection via a triple signal amplification strategy

Chemical Communications ◽

10.1039/d0cc07268k ◽

2021 ◽

Author(s):

Wang He ◽

Bin Qiao ◽

Fengzhen Li ◽

Lisha Pan ◽

Delun Chen ◽

...

Keyword(s):

Heavy Metals ◽

Nucleic Acid ◽

Signal Amplification ◽

Electrochemical Biosensor ◽

Amplification Strategy

We report a novel electrochemical biosensor for the ultrasensitive Hg2+ detection via a signal amplification strategy. This strategy could be easily modified and extended to detect other hazardous heavy metals and nucleic acid.

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