Showing papers on "Nucleic acid methods published in 2021"

Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification.

Abstract: The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.

Integrated Point-of-Care Molecular Diagnostic Devices for Infectious Diseases.

Abstract: The global outbreaks of deadly infectious diseases caused by pathogenic microorganisms have threatened public health worldwide and significantly motivated scientists to satisfy an urgent need for a rapid and accurate detection of pathogens. Traditionally, the culture-based technique is considered as the gold standard for pathogen detection, yet it has a long turnaround time due to the overnight culturing and pathogen isolation. Alternatively, nucleic acid amplification tests provide a relatively shorter turnaround time to identify whether pathogens exist in individuals with high sensitivity and high specificity. In most cases, nucleic acid amplification tests undergo three steps: sample preparation, nucleic acid amplification, and signal transduction. Despite the explosive advancement in nucleic acid amplification and signal transduction technologies, the complex and labor-intensive sample preparation steps remain a bottleneck to create a transformative integrated point-of-care (POC) molecular diagnostic device. Researchers have attempted to simplify and integrate the sample preparations for nucleic acid-based molecular diagnostic devices with innovative progress in integration strategies, engineered materials, reagent storages, and fluid actuation. Therefore, understanding the know-how and obtaining truthful knowledge of existing integrated POC molecular diagnostic devices comprising sample preparations, nucleic acid amplification, and signal transduction can generate innovative solutions to achieve personalized precision medicine and improve global health.In this Account, we discuss the challenges of automated sample preparation solutions integrated with nucleic acid amplification and signal transduction for rapid and precise home diagnostics. Blood, nasal swab, saliva, urine, and stool are emphasized as the most commonly used clinical samples for integrated POC molecular diagnostics of infectious diseases. Even though these five types of samples possess relatively correlated biomarkers due to the human body's circulatory system, each shows unique properties and exclusive advantages for molecular diagnostics in specific situations, which are included in this Account. We examine different integrated POC devices for sample preparation, which includes pathogen isolation and enrichment from the crude sample and nucleic acid purification from isolated pathogens. We present the promising on-chip integration approaches for nucleic acid amplification. We also investigate the on-chip integration methods for reagent storage, which is crucial to simplify the manual operation for end-users. Finally, we present several integrated POC molecular diagnostic devices for infectious diseases. The integrated sample preparation and nucleic acid amplification approach reviewed here can potentially impact the next generation of POC molecular home diagnostic chips, which will significantly impact public health, emergency medicine, and global biosecurity.

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR:

Abstract: Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

A protocol for the detection of genetic markers in saliva by polymerase chain reaction without a nucleic acid purification step: examples of sars-cov-2 and gapdh markers

Abstract: Introduction. Polymerase chain reaction (PCR)-based diagnostic tests use purifi ed nucleic acids (NAs) from clinical samples. The NAs purifi cation step adds time, cost, and aff ects the quality of testing. The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purifi cation of nucleic acids. Methods. PCR, real-time RT-PCR and isothermal amplifi cation tests were used for direct detection of genetic markers, without purifi cation of nucleic acids. Results. We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on a collection of saliva in a solution containing a detergent and ethanol and is compatible with isothermal amplifi cation (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow effi cient detection of external reference and intracellular endogenous markers, while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +4°C or -18°C with the preservation of markers. Storage at room temperature led to the deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by storage at room temperature, provided partial protection. Conclusion. The protocol presented in this report describes the collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests. © 2021 Croatian Veterinary Institute. All rights reserved.

Function, Development and Challenges of COVID-19 Diagnostic Methods in Two Areas: RT-PCR Tests and Serology Tests

Abstract: The ongoing outbreaks of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide pandemic of Coronavirus Disease (COVID-19) in 2019. Nucleic Acid Tests as the current most prevalent method are able to detect the presence of SARS-CoV-2 from infected patients by comparing target viral genome sequences in high sensitivity and accuracy. Three widely applied nucleic acid methods for clinical and research purpose including RT-PCR tests, LAMP and CRISPR-Cas based detection are introduced firstly, followed by the discussion of Antibody Tests, which are ICG and CL immunoassay tests.These two fields of COVID-19 diagnostic methods exhibit some advantages and drawbacks depending on various clinical settings. Antibody test is supplementary and complementary of other diagnostic methods while Nucleic acid tests are overall effectively and rapidly to diagnose infected patients for immediate treatment and isolation. The combination of these two methods may eventually control the dissemination of COVID-19 pandemic. © 2021 The Authors, published by EDP Sciences.

Novel nucleic acid purification chemistry

Abstract: The present invention generally relates to the field of nucleic acid isolation on silica solid support. In particular, a novel silica-solid support nucleic acid binding buffer chemistry is hereby disclosed, which is based on the use of a small quaternary organic compounds, e.g. tetramethylammonium chloride (TMAC), at acidic conditions. This novel nucleic acid purification chemistry purifies not only RNA but also DNA and has the potential for being implementable in a wide variety of commercial kits ranging from the spin columns to integrated Lab-On-A-Chip (LOC) devices such as disposable cartridges that make use of a solid-phase extraction technology. Furthermore, the present methods may be performed using relatively small volumes of binding buffer and consequently in such integrated or closed molecular diagnostic devices, they have the potential of allowing increased volumes of sample input, which for liquid biopsy samples such as plasma or urine, can enhance the chances of detecting rare nucleic acid targets.

Improved rotor mixer for agitation of fluids during sample preparation

Abstract: An apparatus, multi-well plate and method for automated cell lysis and nucleic acid purification and processing. The plate includes a lysis well, at least one wash well, and an elution well. The apparatus includes a vertically aligned rotor mixer comprising a magnetic tip and actuators for moving the rotor mixer in a vertical and horizontal directions, to transfer magnetic beads from well to well. The rotor mixer is used to vortex lysis mixtures, wherein the vortexing speed is sufficient to overcome the magnetic attraction between the beads and mixer tip and disperse the beads in solution, to collect nucleic acids such as DNA.

Nucleic acid purification method and nucleic acid purification equipment

Abstract: The invention provides a nucleic acid purification method and nucleic acid purification equipment, and relates to the technical field of biology. The nucleic acid purification method provided by the invention comprises the following steps: firstly, mixing a sample with a lysis solution and magnetic beads, so that nucleic acid after cell lysis is fully adsorbed by the magnetic beads; and then, intermittently moving a magnetic bead collecting device to the bottom from the liquid level of a cracked solution, so that the operation mode of the magnetic bead collecting device cooperates with the sedimentation of the magnetic beads, the magnetic beads are fully adsorbed in an extremely short time, and the high-efficiency purification of the nucleic acid is further realized. The purification method disclosed by the invention is simple, quick and efficient, can finish extraction and purification of the nucleic acid within 8 minutes, and is high in extraction efficiency.