Ultrasensitive and Whole-Course Encapsulated Field Detection of 2019-nCoV Gene Applying Exponential Amplification from RNA Combined with Chemical Probes

10.26434/chemrxiv.12012789 ◽

2020 ◽

Cited By ~ 1

Author(s):

Simin Xia ◽

Xi Chen

Keyword(s):

Reverse Transcription ◽

Rna Virus ◽

Detection Methods ◽

Chemical Probes ◽

Trace Level ◽

Field Detection ◽

Global Epidemic ◽

Affinity Probe ◽

Highly Sensitive ◽

Human Infections

The newly identified 2019-nCoV (SARS-CoV-2) RNA virus has caused over 80,000 laboratory-confirmed human infections in China (as of Mar. 1st, 2020), and is now becoming a global epidemic issue. As a result, highly sensitive, reliable and field-deployable methods to detect 2019-nCoV that can be developed in a shortest possible time are urgently needed. In this work, we introduce whole-course encapsulated and ultrasensitive field detection methods against 2019-nCoV gene. We applied isothermal exponential amplification methods via reverse transcription plus subsequent enzymatic recombinase amplification (ERA), a modified recombinase polymerase amplification (RPA), to amplify trace level of 2019-nCoV RNA. We designed both exo FRET probe and nfo affinity probe for easy detection of the amplified nucleic acids. These methods can be developed within a few days and as low as 0.32 aM (i.e. 0.32×10-18 M) of RNA can be reliably detected.<b></b><br>

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RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

Diagnostics ◽

10.3390/diagnostics11091646 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1646

Author(s):

Kasturi Selvam ◽

Mohamad Ahmad Najib ◽

Muhammad Fazli Khalid ◽

Suharni Mohamad ◽

Fahreddin Palaz ◽

...

Keyword(s):

Reverse Transcription ◽

Quantitative Polymerase Chain Reaction ◽

Diagnostic Techniques ◽

Diagnostic Methods ◽

Detection Methods ◽

Molecular Diagnostic ◽

Public Attention ◽

Laboratory Equipment ◽

Highly Sensitive ◽

Polymerase Chain

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has attracted public attention. The gold standard for diagnosing COVID-19 is reverse transcription–quantitative polymerase chain reaction (RT-qPCR). However, RT-qPCR can only be performed in centralized laboratories due to the requirement for advanced laboratory equipment and qualified workers. In the last decade, clustered regularly interspaced short palindromic repeats (CRISPR) technology has shown considerable promise in the development of rapid, highly sensitive, and specific molecular diagnostic methods that do not require complicated instrumentation. During the current COVID-19 pandemic, there has been growing interest in using CRISPR-based diagnostic techniques to develop rapid and accurate assays for detecting SARS-CoV-2. In this work, we review and summarize reverse-transcription loop-mediated isothermal amplification (RT-LAMP) CRISPR-based diagnostic techniques for detecting SARS-CoV-2.

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Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection

Analytical Chemistry ◽

10.1021/acs.analchem.1c00013 ◽

2021 ◽

Author(s):

Wei S. Zhang ◽

Jianbin Pan ◽

Feng Li ◽

Min Zhu ◽

Mengting Xu ◽

...

Keyword(s):

Reverse Transcription ◽

Recombinase Polymerase Amplification ◽

Highly Sensitive

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Detection by real-time quaking-induced conversion (RT-QuIC), ELISA, and IHC of chronic wasting disease prion in lymph nodes from Pennsylvania white-tailed deer with specific PRNP genotypes

Journal of Veterinary Diagnostic Investigation ◽

10.1177/10406387211021411 ◽

2021 ◽

pp. 104063872110214

Author(s):

Deepanker Tewari ◽

David Steward ◽

Melinda Fasnacht ◽

Julia Livengood

Keyword(s):

Real Time ◽

Disease Susceptibility ◽

Chronic Wasting Disease ◽

Low Frequency ◽

The United States ◽

Detection Methods ◽

Spongiform Encephalopathy ◽

Wasting Disease ◽

Diagnostic Laboratory ◽

Highly Sensitive

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer ( Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

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A rapid and accurate method for screening T-2 toxin in food and feed using competitive AlphaLISA

FEMS Microbiology Letters ◽

10.1093/femsle/fnab029 ◽

2021 ◽

Vol 368 (6) ◽

Author(s):

Liwen Zhang ◽

Qingyu Lv ◽

Yuling Zheng ◽

Xuan Chen ◽

Decong Kong ◽

...

Keyword(s):

High Sensitivity ◽

Accurate Method ◽

Detection Methods ◽

Cereal Crops ◽

Detection Range ◽

Highly Sensitive ◽

Food And Feed ◽

Good Repeatability ◽

Feed Samples ◽

Better Than

ABSTRACT T-2 is a common mycotoxin contaminating cereal crops. Chronic consumption of food contaminated with T-2 toxin can lead to death, so simple and accurate detection methods in food and feed are necessary. In this paper, we establish a highly sensitive and accurate method for detecting T-2 toxin using AlphaLISA. The system consists of acceptor beads labeled with T-2-bovine serum albumin (BSA), streptavidin-labeled donor beads and biotinylated T-2 antibodies. T-2 in the sample matrix competes with T-2-BSA for antibodies. Adding biotinylated antibodies to the test well followed by T-2 and T-2-BSA acceptor beads yielded a detection range of 0.03–500 ng/mL. The half-maximal inhibitory concentration was 2.28 ng/mL and the coefficient of variation was <10%. In addition, this method had no cross-reaction with other related mycotoxins. This optimized method for extracting T-2 from food and feed samples achieved a recovery rate of approximately 90% in T-2 concentrations as low as 1 ng/mL, better than the performance of a commercial ELISA kit. This competitive AlphaLISA method offers high sensitivity, good specificity, good repeatability and simple operation for detecting T-2 toxin in food and feed.

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