scholarly journals Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP

Lab on a Chip ◽  
2019 ◽  
Vol 19 (6) ◽  
pp. 1035-1040 ◽  
Author(s):  
Jason E. Kreutz ◽  
Jiasi Wang ◽  
Allison M. Sheen ◽  
Alison M. Thompson ◽  
Jeannette P. Staheli ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Sensitivity And Specificity ◽  
Absolute Quantification ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Quantitative Detection

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids.

scholarly journals Use of Microfluidic Assays to Develop Reliable and Economic Nucleic Acid Application Technologies, Employing MicroRNAs for the Diagnostic Screening of Colon Cancer in Human Stool in Low-Resource Settings

2020 ◽  
Vol 5 (4) ◽  
pp. 1-29
Author(s):  
Farid E. Ahmed ◽  
Mostafa M. Gouda ◽  
Nancy C. Ahmed
Keyword(s):  
Colon Cancer ◽  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Rolling Circle Amplification ◽  
Nucleic Acid Amplification ◽  
Performance Criteria ◽  
Quantitative Detection ◽  
Diagnostic Screening ◽  
Low Resource ◽  
Microfluidic Technology

Isolation methods that employ readily-available inexpensive supplies on the open market, which are reliable, as well as economical, such as nucleic acid amplification techniques (NAAT) based on microfluidic technology in low-resource research settings (LRRS) that meets the ASSURED guidelines are essential to develop a noninvasive diagnostic colon cancer screen in stool using micro(mi)RNA molecules. A combination of a microfluidic-based MiRNA stool test with a reliable rolling circle amplification/detection method applied to the quantification of miRNA molecules, result in an affordable sensitive and specific isothermal method for the noninvasive quantitative detection of miRNAs in LRRS. Scientists and engineers have become interested in miRNAs, and they have intensified their efforts to apply emerging simple detection tools to the important bioanalytical challenge of quantifying these small 18-26 nt long molecules. Some of the proposed approaches incorporate novel material, such as simple centrifuges and methods based on microfluidic technology, while others utilize the interesting biological properties of these molecules, such as forming branched RCA structures, allowing for the detection of these biomarker molecules at an attomolar "aM" concentration level, using low cost extraction and isothermal amplification methods in LRRS. We have been interested in studying colorectal cancer (CRC) because it is the 3rd most common malignancy worldwide, and stool can be obtained noninvasively from the patients. We have focused in this research on colon cancer (CC) because it is more common in the USA than rectal cancer (RC). The innovation of our approach lies in the exploratory use of an affordable, quantitative miRNA profiling in noninvasive stool samples in LRRS, whose extracted fragile total RNA is stabilized shortly after excretion from stool by commercially available kits, so it does not ever fragment, followed by quantitative standardized analytical tests that are neither labor intensive, nor require expensive instrumentation, in order to develop apanel of novel miRNA genes for the noninvasive diagnostic screening of early left and right sporadic colon cancers, more economically, and with higher sensitivity and specificity than any other colon cancer screening test currently available on the market. To show the clinical sensitivity and specificity of the proposed quantitative miRNA test using simple methodologies in LRRS,the miRNA results are to be correlated with FOBT, colonoscopy, and pathology data. Standardization establishes test’s performance criteria (sample selection, optimal sample running conditions, preservation and storage), in order to ensure that the assay will perform the same way in any laboratory, by any trained personnel, anywhere in low-resource laboratory settings worldwide.

scholarly journals Use of Microfluidic Assays to Develop Reliable and Economic Nucleic Acid Application Technologies, Employing MicroRNAs for the Diagnostic Screening of Colon Cancer in Human Stool in Low-Resource Settings

2020 ◽  
Vol 6 (1) ◽  
pp. 44-72
Author(s):  
Farid E. Ahmed ◽  
Mostafa M. Gouda ◽  
Nancy C. Ahmed
Keyword(s):  
Colon Cancer ◽  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Rolling Circle Amplification ◽  
Nucleic Acid Amplification ◽  
Performance Criteria ◽  
Quantitative Detection ◽  
Diagnostic Screening ◽  
Low Resource ◽  
Microfluidic Technology

Isolation methods that employ readily-available inexpensive supplies on the open market, which are reliable, as well as economical, such as nucleic acid amplification techniques (NAAT) based on microfluidic technology in low-resource research settings (LRRS) that meets the ASSURED guidelines are essential to develop a noninvasive diagnostic colon cancer screen in stool using micro(mi)RNA molecules. A combination of a microfluidic-based MiRNA stool test with a reliable rolling circle amplification/detection method applied to the quantification of miRNA molecules, result in an affordable sensitive and specific isothermal method for the noninvasive quantitative detection of miRNAs in LRRS. Scientists and engineers have become interested in miRNAs, and they have intensified their efforts to apply emerging simple detection tools to the important bioanalytical challenge of quantifying these small 18-26 nt long molecules. Some of the proposed approaches incorporate novel material, such as simple centrifuges and methods based on microfluidic technology, while others utilize the interesting biological properties of these molecules, such as forming branched RCA structures, allowing for the detection of these biomarker molecules at an attomolar "aM" concentration level, using low cost extraction and isothermal amplification methods in LRRS. We have been interested in studying colorectal cancer (CRC) because it is the 3rd most common malignancy worldwide, and stool can be obtained noninvasively from the patients. We have focused in this research on colon cancer (CC) because it is more common in the USA than rectal cancer (RC). The innovation of our approach lies in the exploratory use of an affordable, quantitative miRNA profiling in noninvasive stool samples in LRRS, whose extracted fragile total RNA is stabilized shortly after excretion from stool by commercially available kits, so it does not ever fragment, followed by quantitative standardized analytical tests that are neither labor intensive, nor require expensive instrumentation, in order to develop apanel of novel miRNA genes for the noninvasive diagnostic screening of early left and right sporadic colon cancers, more economically, and with higher sensitivity and specificity than any other colon cancer screening test currently available on the market. To show the clinical sensitivity and specificity of the proposed quantitative miRNA test using simple methodologies in LRRS,the miRNA results are to be correlated with FOBT, colonoscopy, and pathology data. Standardization establishes test’s performance criteria (sample selection, optimal sample running conditions, preservation and storage), in order to ensure that the assay will perform the same way in any laboratory, by any trained personnel, anywhere in low-resource laboratory settings worldwide.

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 Analytical and Clinical Comparison of Three Nucleic Acid Amplification Tests for SARS-CoV-2 Detection

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Elizabeth Smith ◽  
Wei Zhen ◽  
Ryhana Manji ◽  
Deborah Schron ◽  
Scott Duong ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Drug Administration ◽  
Clinical Performance ◽  
Absolute Quantification ◽  
Nucleic Acid Amplification ◽  
Clinical Correlation ◽  
Molecular Assays ◽  
Percent Agreement ◽  
Rna Transcript

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in December 2019 and has quickly become a worldwide pandemic. In response, many diagnostic manufacturers have developed molecular assays for SARS-CoV-2 under the Food and Drug Administration (FDA) Emergency Use Authorization (EUA) pathway. This study compared three of these assays, the Hologic Panther Fusion SARS-CoV-2 assay (Fusion), the Hologic Aptima SARS-CoV-2 assay (Aptima), and the BioFire Defense COVID-19 test (BioFire), to determine analytical and clinical performance as well as workflow. All three assays showed similar limits of detection (LODs) using inactivated virus, with 100% detection, ranging from 500 to 1,000 genome equivalents/ml, whereas use of a quantified RNA transcript standard showed the same trend but had values ranging from 62.5 to 125 copies/ml, confirming variability in absolute quantification of reference standards. The clinical correlation found that the Fusion and BioFire assays had a positive percent agreement (PPA) of 98.7%, followed by the Aptima assay at 94.7%, compared to the consensus result. All three assays exhibited 100% negative percent agreement (NPA). Analysis of discordant results revealed that all four samples missed by the Aptima assay had cycle threshold (Ct) values of >37 by the Fusion assay. In conclusion, while all three assays showed similar relative LODs, we showed differences in absolute LODs depending on which standard was employed. In addition, the Fusion and BioFire assays showed better clinical performance, while the Aptima assay showed a modest decrease in overall PPA. These findings should be kept in mind when making platform testing decisions.

Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

1995 ◽  
Vol 58 (12) ◽  
pp. 1357-1362 ◽  
Author(s):  
LEE-ANN JAYKUS ◽  
RICARDO DE LEON ◽  
MARK D. SOBSEY
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Ammonium Bromide ◽  
Rt Pcr ◽  
Initial Inoculum ◽  
Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

Nucleic acid amplification-based methods for microRNA detection

2015 ◽  
Vol 7 (6) ◽  
pp. 2258-2263 ◽  
Author(s):  
Hui-Ling Chen ◽  
Meng-Meng Guo ◽  
Hao Tang ◽  
Zhan Wu ◽  
Li-Juan Tang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Basic Principles ◽  
Microrna Detection

This review traces the basic principles of several nucleic acid amplification-based microRNA detection methods that have been developed in recent three years.

scholarly journals Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

2020 ◽  
Author(s):  
Soon Keong Wee ◽  
Suppiah Paramalingam Sivalingam ◽  
Eric Peng Huat Yap
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
N Gene ◽  
Direct Pcr ◽  
Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

scholarly journals Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

PLoS ONE ◽  
2010 ◽  
Vol 5 (11) ◽  
pp. e14155 ◽  
Author(s):  
Olga A. Gandelman ◽  
Vicki L. Church ◽  
Cathy A. Moore ◽  
Guy Kiddle ◽  
Christopher A. Carne ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Nucleic Acid Amplification ◽  
Quantitative Detection

Empfehlungen zur qualitätsgesicherten Durchführung von Nukleinsäure-Amplifikations - Testungen (NAT) von Blutprodukten. Quality assurance in nucleic acid amplification testing (NAT) of blood products

2005 ◽  
Vol 29 (2) ◽  
Author(s):  
Willi K. Roth
Keyword(s):  
Quality Control ◽  
Nucleic Acid ◽  
Sample Preparation ◽  
Nucleic Acids ◽  
Internal Control ◽  
Control Measures ◽  
Nucleic Acid Amplification ◽  
Blood Products ◽  
Whole Process ◽  
And Storage

AbstractEuropean manufacturers of plasma products and German blood transfusion services were the first to introduce nucleic acid amplification testing (NAT) of blood products in the mid-1990s. Their primary goal was to increase the safety of blood by closing as far as possible the diagnostic window, which exists after the onset of viral infection until the appearance of the first detectable antibodies. Sample preparation, transport and storage are crucial steps in a quality-controlled PCR. Sensitivity and contamination rates highly depend on the sample preparation and storage techniques. Anticoagulants must be selected carefully because some may inhibit the PCR. Dilution of samples by pooling needs to be considered and should be compensated for by subsequent virus enrichment procedures, e.g. centrifugation. The whole process of sample preparation, pooling and virus enrichment must be validated and quality control measures must be implemented. Reagents for the extraction of viral nucleic acids should not pose any risk to the laboratory staff. Nevertheless, the reagents should be highly efficient in liberating viral nucleic acids at high yield and purity for the following amplification reactions. At this critical stage, quality control measures should guarantee an efficient extraction process and contain potential sources of contaminations. Several methods are available for the amplification of nucleic acids. PCR is the most common, especially in in-house assays. The amplification of nucleic acids should be performed as far as possible in a closed system, which may be guaranteed best by real-time PCR approaches. Reaction tubes need never be opened during the amplification because detection can be performed through the closed tube. Amplicons that could contaminate the following PCR reactions will not be released. It is of great importance to blood transfusion services to guarantee that negative results un-equivocally indicate virus negative blood donations. Therefore, internal control sequences should be implemented in each individual PCR reaction in order to monitor that the individual PCR has worked correctly. Besides internal control sequences, external negative and positive controls should be implemented in each PCR run to demonstrate false positive reactions as well as to monitor pre-PCR processes like virus enrichment and extraction. The whole process needs to be validated according to the criteria set in national guidelines or by national authorities. External quality assessment programs are highly recommended.

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