scholarly journals Engineered Janus probes modulate nucleic acid amplification to expand the dynamic range for direct detection of viral genomes in one microliter crude serum samples

2018 ◽  
Vol 9 (2) ◽  
pp. 392-397 ◽  
Author(s):  
Yue Zhao ◽  
Feng Chen ◽  
Jing Qin ◽  
Jing Wei ◽  
Wenhua Wu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Viral Genome ◽  
Dynamic Range ◽  
Direct Detection ◽  
Nucleic Acid Amplification ◽  
Serum Samples ◽  
Viral Genomes

Janus probes were designed to expand the dynamic range of amplification for viral genome quantification in 1 μL crude serum.

scholarly journals Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration

2019 ◽  
Vol 116 (33) ◽  
pp. 16240-16249 ◽  
Author(s):  
Wei Ouyang ◽  
Jongyoon Han
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Molecular Diagnostics ◽  
Troponin I ◽  
Direct Detection ◽  
Signal To Noise Ratio ◽  
Protein Detection ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Enzyme Linked Immunosorbent Assay

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.

scholarly journals CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

2019 ◽  
Author(s):  
Richard Bruch ◽  
Julia Baaske ◽  
Claire Chatelle ◽  
Mailin Meirich ◽  
Sibylle Madlener ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Early Stage ◽  
Low Cost ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Process Time ◽  
Serum Samples ◽  
Microfluidic Biosensor ◽  
Pcr Method

Non-coding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated to many different diseases, such as cancer, dementia or cardiovascular conditions. The key for an effective treatment is an accurate initial diagnosis at an early stage, improving the patient’s survival chances. Here, we introduce a CRISPR/Cas13a powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a has been realized without any nucleic acid amplification. With a readout time of 9 minutes and an overall process time of less than 4 hours, a limit of detection of 10 pM was achieved, using a measuring volume of less than 0.6 µl. Furthermore, we demonstrate the feasibility of our versatile sensor platform to detect miR-19b in serum samples of children, suffering from brain cancer. The validation of our results with a standard qRT-PCR method shows the ability of our system to be a low-cost and target amplification-free tool for nucleic acid based diagnostics.

scholarly journals A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care

2020 ◽  
Author(s):  
Diem Hong Tran ◽  
Hoang Quoc Cuong ◽  
Hau Thi Tran ◽  
Uyen Phuong Le ◽  
Hoang Dang Khoa Do ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
Nucleic Acid Amplification ◽  
Gold Standard Method ◽  
Synthetic Dna ◽  
Isothermal Nucleic Acid Amplification ◽  
Freeze Dried ◽  
Test Kit

ABSTRACTThe COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-to-use kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon

Talanta ◽  
2016 ◽  
Vol 154 ◽  
pp. 520-525 ◽  
Author(s):  
Zhen Gui ◽  
Quanbo Wang ◽  
Jinchang Li ◽  
Mingchen Zhu ◽  
Lili Yu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Nucleic Acid ◽  
Molecular Beacon ◽  
Direct Detection ◽  
Locked Nucleic Acid ◽  
Serum Samples ◽  
Circulating Free Dna ◽  
Free Dna

scholarly journals Comparison between TRCReady MTB and MTB ELITe MGB kit for the direct detection of Mycobacterium tuberculosis complex in respiratory and non-respiratory samples

2017 ◽  
Vol 32 (2) ◽  
Author(s):  
Arianna Gatti ◽  
Paolo Melloni ◽  
Emanuela Vasconi ◽  
Carlo Agrappi ◽  
Paola Mirri ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Direct Detection ◽  
Nucleic Acid Amplification ◽  
World Health ◽  
Negative Results ◽  
Nucleic Acid Amplification Assay ◽  
Amplification Assay ◽  
Health Organization ◽  
Analytical Time ◽  
Respiratory Specimens

<em>Background</em>: A recent World Health Organization survey estimated about 10.4 million new tuberculosis (TB) cases per year with a high mortality rate. A rapid and accurate diagnosis of TB is very important for the optimal treatment and the prevention of spread. In the last few years rapid nucleic acid amplification tests for the detection of <em>Mycobacterium</em> <em>tuberculosis</em> <em>complex</em> (MTB) were developed. <br /><em>Materials and methods</em>: In this study we have compared TRCReady MTB, based on the Transcription Reverse Transcription Concerted Reaction technology (TRC) and MTB ELITe MGB Kit, a qualitative nucleic acid amplification assay, for the direct detection of MTB in 56 respiratory and 24 non-respiratory specimens, collected from September to November 2015. <br /><em>Results</em>: Both methods did not detect any MTB in 45 respiratory samples and in 22 non-respiratory specimens with negative cultures. MTB ELITe MGB Kit identified MTB in 8 respiratory samples with MTB positive cultures, 7 of which were detected by TRCReady MTB as well. Two non-respiratory MTB positive cultures were correctly identified by both methods. In two respiratory samples with <em>Mycobacterium Other Than Tuberculosis</em> positive cultures both methods provided negative results. <br /><em>Conclusions</em>: In conclusion, TRCReady MTB performance proved comparable to that of MTB ELITe MGB Kit in the diagnosis of pulmonary and extra-pulmonary TB, with a shorter analytical time (50 vs 110 min).

scholarly journals Nucleic Acid-Based Sensing Techniques for Diagnostics and Surveillance of Influenza

Biosensors ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 47 ◽  
Author(s):  
Samantha J. Courtney ◽  
Zachary R. Stromberg ◽  
Jessica Z. Kubicek-Sutherland
Keyword(s):  
Influenza Virus ◽  
Nucleic Acid ◽  
Diagnostic Test ◽  
Influenza A ◽  
Direct Detection ◽  
Point Of Care ◽  
Influenza Viruses ◽  
Nucleic Acid Amplification ◽  
High Genetic Diversity ◽  
Mild Disease

Influenza virus poses a threat to global health by causing seasonal outbreaks as well as three pandemics in the 20th century. In humans, disease is primarily caused by influenza A and B viruses, while influenza C virus causes mild disease mostly in children. Influenza D is an emerging virus found in cattle and pigs. To mitigate the morbidity and mortality associated with influenza, rapid and accurate diagnostic tests need to be deployed. However, the high genetic diversity displayed by influenza viruses presents a challenge to the development of a robust diagnostic test. Nucleic acid-based tests are more accurate than rapid antigen tests for influenza and are therefore better candidates to be used in both diagnostic and surveillance applications. Here, we review various nucleic acid-based techniques that have been applied towards the detection of influenza viruses in order to evaluate their utility as both diagnostic and surveillance tools. We discuss both traditional as well as novel methods to detect influenza viruses by covering techniques that require nucleic acid amplification or direct detection of viral RNA as well as comparing advantages and limitations for each method. There has been substantial progress in the development of nucleic acid-based sensing techniques for the detection of influenza virus. However, there is still an urgent need for a rapid and reliable influenza diagnostic test that can be used at point-of-care in order to enhance responsiveness to both seasonal and pandemic influenza outbreaks.

scholarly journals Evaluation of Three Nucleic Acid Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

1999 ◽  
Vol 37 (6) ◽  
pp. 1932-1934 ◽  
Author(s):  
S. X. Wang ◽  
L. Tay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Mycobacterium Tuberculosis Complex ◽  
Direct Detection ◽  
Nucleic Acid Amplification ◽  
Smear Microscopy ◽  
Direct Test ◽  
Tuberculosis Complex ◽  
Chain Reaction ◽  
Respiratory Specimens

Two hundred thirty respiratory specimens from 230 patients were analyzed by using COBAS AMPLICOR PCR, Amplified Mycobacterium tuberculosis Direct Test, and ligase chain reaction methods. Results were compared with those of smear microscopy and radiometric culture (Bactec) methods. No significant differences were observed among the results of the three methods, which are acceptable for direct detection of M. tuberculosis complex in respiratory specimens.

Performance and Utilization of a Laboratory-Developed Nucleic Acid Amplification Test (NAAT) for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low-Prevalence Area

2020 ◽  
Vol 154 (1) ◽  
pp. 115-123
Author(s):  
Sanchita Das ◽  
Kathy A Mangold ◽  
Nirav S Shah ◽  
Lance R Peterson ◽  
Richard B Thomson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Direct Detection ◽  
Bacterial Load ◽  
Extrapulmonary Tuberculosis ◽  
Nucleic Acid Amplification ◽  
Benefit Patient ◽  
Smear Negative ◽  
Low Prevalence ◽  
Culture Positive ◽  
Global Health Problem

Abstract Objectives Tuberculosis (TB) is a significant global health problem. In low-prevalence areas and low clinical suspicion, nucleic acid amplification tests (NAAT) for direct detection of Mycobacterium tuberculosis complex (MTBC) can speed therapy initiation and infection control. An NAAT assay (TBPCR) targeting MTBC IS6110 is used for detecting MTBC in our low-prevalence population. Methods Fifteen-year review of patient records identified 146 patients with culture-positive pulmonary tuberculosis (PTB) or extrapulmonary tuberculosis (EPTB). Laboratory-developed TBPCR was retrospectively compared with standard stain and cultures for PTB and EPTB diagnoses. Results TBPCR assay was used in 57% of patients with PTB and 33% of patients with EPTB. TBPCR detected 88.4% of all TB (smear-positive, 97%; smear-negative, 79%) with 100% specificity. Low bacterial load was indicated in TBPCR-negative PTB (P = .002) and EPTB (P &lt; .008). Conclusions TBPCR performance was optimum but significantly underused. Guidelines are proposed for mandated use of TBPCR that capture patients with clinically suspected PTB. Focused TBPCR use in low prevalence populations will benefit patient care, infection prevention, and public health.

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