scholarly journals Inactivating porcine coronavirus before nuclei acid isolation with the temperature higher than 56 °C damages its genome integrity seriously

2020 ◽  
Author(s):  
Qingxin Zhang ◽  
Qingshun Zhao
Keyword(s):  
High Temperature ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Virus Disease ◽  
Genome Integrity ◽  
Nucleic Acid Detection ◽  
Chinese Society ◽  
Diarrhea Virus ◽  
The Impact ◽  
Temperature Inactivation

Abstract2019-Novel Coronavirus (2019-nCoV) is the pathogen of Corona Virus Disease 2019. Nucleic acid detection of 2019-nCoV is one of the key indicators for clinical diagnosis. However, the positive rate is only 30-50%. Currently, fluorescent quantitative RT-PCR technology is mainly used to detect 2019-nCoV. According to “The Laboratory Technical Guidelines for Detection 2019-nCoV (Fourth Edition)” issued by National Health and Commission of China and “The Experts’ Consensus on Nucleic Acid Detection of 2019-nCoV” released by Chinese Society of Laboratory Medicine, the human samples must be placed under 56°C or higher to inactivate the viruses in order to keep the inspectors from virus infection before the nucleic acids were isolated as the template of qRT-PCR. In this study, we demonstrated that the virus inactivation treatment disrupts its genome integrity seriously when using porcine epidemic diarrhea virus (vaccine), a kind of coronavirus, as a model. Our results showed that only 50.11% of the detectable viral templates left after the inactivation of 56 °C for 30 minutes and only 3.36% left after the inactivation of 92 °C for 5 minutes when the samples were preserved by Hank’s solution, one of an isotonic salt solutions currently suggested. However, the detectable templates of viral nucleic acids can be unchanged after the samples were incubated at 56 °C or higher if the samples were preserved with an optimized solution to protect the RNA from being disrupted. We therefore highly recommend to carry out systematic investigation on the impact of high temperature inactivation on the integrity of 2019-nCoV genome and develop a sample preservation solution to protect the detectable templates of 2019-nCoV nucleic acids from high temperature inactivation damage.

scholarly journals Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander S. Day ◽  
Tiffany-Heather Ulep ◽  
Elizabeth Budiman ◽  
Laurel Dieckhaus ◽  
Babak Safavinia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Light Scatter ◽  
Emulsion Droplets ◽  
Assay Performance ◽  
Bacterial Adsorption ◽  
Scatter Intensity ◽  
The Impact ◽  
Theory Light

AbstractAn emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

The application of viral nucleic acid detection and lung CT in COVID-19 diagnosis

2020 ◽  
Author(s):  
Rui Hu
Keyword(s):  
Nucleic Acid ◽  
Clinical Symptoms ◽  
Virus Disease ◽  
False Negative ◽  
False Negative Rate ◽  
Diagnostic Methods ◽  
Nucleic Acid Detection ◽  
Viral Nucleic Acid ◽  
Early Medical Intervention ◽  
Lung Ct

The Corona Virus Disease 2019 (COVID-19) has the characteristics of fast propagation speed and strong pathogenicity and has attracted wide attention of people, medical workers, and researchers around the world. Accurate, rapid, and timely screening and diagnosis of COVID-19 is of great significance to control the development of the epidemic situation and save the lives of patients. Currently, the detection of viral nucleic acid and lung CT is the main screening and diagnostic methods of COVID-19. Nucleic acid detection has the advantages of fast, strong specificity and high sensitivity, but there is a certain false-negative rate. CT result of lung examination is visual, but it is not typical due to the uncertain time of clinical symptoms and the early medical intervention. Therefore, the diagnosis of COVID-19 should include a combination of epidemiological history, clinical symptoms, imaging, and laboratory tests.

scholarly journals A nuclease protection ELISA assay for colorimetric and electrochemical detection of nucleic acids

2019 ◽  
Vol 11 (8) ◽  
pp. 1027-1034 ◽  
Author(s):  
Jessica E. Filer ◽  
Robert B. Channon ◽  
Charles S. Henry ◽  
Brian J. Geiss
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Electrochemical Detection ◽  
Nucleic Acid Detection ◽  
Elisa Assay

The NP-ELISA combines traditional nuclease protection with optical and electrochemical enzymatic readout for nucleic acid detection.

Recent advance in exonuclease Ⅲ-assisted target signal amplification strategy for nucleic acid detection

2021 ◽  
Author(s):  
hongyu liu ◽  
Yuhao You ◽  
Youzhuo Zhu ◽  
Heng Zheng
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Molecular Diagnostics ◽  
Mutation Analysis ◽  
Signal Amplification ◽  
Nucleic Acid Detection ◽  
Target Signal ◽  
Forensic Investigations ◽  
Exo Iii ◽  
Amplification Strategy

Detection of nucleic acids have become significantly important in molecular diagnostics, genetics therapy, mutation analysis, forensic investigations and biomedical development, and so on. In recent years, exonuclease Ⅲ (Exo III)...

A catalytic DNA circuit-programmed and enzyme-powered autonomous DNA machine for nucleic acid detection

The Analyst ◽  
2019 ◽  
Vol 144 (20) ◽  
pp. 5923-5927 ◽  
Author(s):  
Shuang Liu ◽  
Chen Xin ◽  
Xiaoxiao Yu ◽  
Zhenbo Ding ◽  
Shufeng Liu
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Detection ◽  
One Step ◽  
Catalytic Dna ◽  
Dna Machine

A catalytic DNA circuit-programmed and enzyme-powered autonomous DNA machine was proposed for one-step, isothermal and dual-level amplified detection of nucleic acids.

NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification

Lab on a Chip ◽  
2015 ◽  
Vol 15 (7) ◽  
pp. 1697-1707 ◽  
Author(s):  
Mark D. Borysiak ◽  
Kevin W. Kimura ◽  
Jonathan D. Posner
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Mobile Phone ◽  
Isothermal Amplification ◽  
Nucleic Acid Detection ◽  
Complex Matrices ◽  
Loop Mediated Isothermal Amplification

The NAIL device integrates isotachophoresis and loop-mediated isothermal amplification (LAMP) with mobile phone detection to extract, amplify, and detect nucleic acids from complex matrices in less than one hour.

scholarly journals DNA extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid

2020 ◽  
Author(s):  
Erika Ganda ◽  
Kristen L. Beck ◽  
Niina Haiminen ◽  
Ban Kawas ◽  
Brittany Cronk ◽  
...  
Keyword(s):  
Food Safety ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Biological Fluid ◽  
Genetic Material ◽  
Bovine Milk ◽  
Extraction Methods ◽  
Acid Extraction ◽  
Primary Material ◽  
The Impact

ABSTRACTAbstractUntargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis could reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering primary material, laboratory methods, data analysis, and interpretation.The focus of this study is in gathering the primary material, in this case, DNA. We used bovine milk as a model to 1) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk; 2) evaluate host DNA depletion methods for use with milk, and 3) develop and evaluate a selective lysis-PMA based protocol for host DNA depletion in milk.Our results suggest that magnetic-based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety where the detection of Gram-negative pathogens and indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host.ImportanceTracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of what the relative composition looks like. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around one thousand times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods in the ability of detecting specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

Study of dielectrophoresis effect of dipolar molecules in electric field of nanopore and development of medical diagnostic nanopore sensor

2015 ◽  
Author(s):  
◽  
Kai Tian
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Early Stage ◽  
Medical Diagnostics ◽  
Nucleic Acid Detection ◽  
Detection Accuracy ◽  
University Of Missouri ◽  
Complex Samples ◽  
Medical Diagnostic ◽  
Polycationic Peptide

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The nanopore sensor can detect cancer-derived nucleic acid biomarkers such as microRNAs (miRNAs), providing a noninvasive tool potentially useful in medical diagnostics. However, the nanopore-based detection of these biomarkers remains confounded by the presence of numerous other nucleic acid species found in biofluid extracts. Their nonspecific interactions with the nanopore inevitably contaminate the target signals, reducing the detection accuracy. Here we report a novel method that utilizes a polycationic peptide-PNA probe as the carrier for selective nucleic acid detection in the nucleic acids mixture. The cationic probe hybridized with DNA or RNA forms a dipole complex, which can be captured by the pore using a voltage polarity that is opposite the polarity used to capture negatively charged nucleic acids. As a result, non-target species are driven away from the pore opening, and the target sequences can be detected accurately without interference. In addition, we demonstrate that the PNA probe enables to accurately discriminate single-nucleotide difference. Moreover, molecule dynamic simulation is applied to expose the mechanism. Combined with experimental and calculating data, we construct a model to demonstrate that it is universal for all kinds of nucleic acid targets. In sum, this highly sensitive and selective nano-dielectrophoresis approach can be applied to the detection of clinically relevant nucleic acid fragments in complex samples and fulfills the diagnostic of diseases in early stage.

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